Autoantibodies and their targets in the diagnosis of peripheral neuropathies

ABSTRACT

The present invention relates to methods of aiding in the diagnosis of peripheral neuropathies that comprise determining the titer of autoantibodies directed toward particular nervous system antigens. It also provides for substantially purified preparations of specific antigens, namely neuroprotein-1, neuroprotein-2, neuroprotein-3, neuroprotein-4 and neuroprotein-5, which may be used in such diagnostic methods.

This invention was made with government support under grant number AG07438 awarded by the National Institutes of Health. The government hascertain rights in the invention.

This application is a continuation of U.S. patent application No.07/743,005, filed Aug. 9, 1991, now abandoned.

TABLE OF CONTENTS

1. Introduction

2. Background Of The Invention

2.1. Peripheral Neuropathies

2.1.1. Amyotrophic Lateral Sclerosis

2.1.2. Multifocal Motor Neuropathy With Conduction Block

2.1.3. Sensory Neuropathies

2.2. Antigenic Structures Of The Peripheral Nervous System

2.2.1. Gangliosides

2.2.2. Myelin-Associated Glycoprotein

2.3. Antibodies In Peripheral Neuropathies

2.3.1. Anti-GM₁ Antibodies

2.3.2. Anti-MAG Antibodies

3. Summary Of The Invention

4. Description Of The Figures

5. Detailed Description Of The Invention

5.1. Characterization Of Antigens

5.2. Methods Of Diagnosis Of Peripheral Neuropathies

5.3. Preparation Of Antibodies

5.4. Additional Utilities Of The Invention

6. Example: Polyneuropathy Syndrome Associated With Serum Antibodies ToSulfatide And Myelin-Associated Glycoprotein

6.1. Materials And Methods

6.1.1. Patients

6.1.2. ELISA Antibody Assays

6.1.3. Immunoblot Assays

6.1.4. Immunostaining After High-Performance Thin-Layer Chromatography(HPTLC)

6.2. Results

6.2.1. Serum Antibody Testing

6.2.2. Correlations Between Antibody Reactivity And Clinical AndPhysiological Patterns

6.3. Discussion

6.3.1. Patients With Anti-Sulfatide Antibodies

6.3.2. Patients With IgM Anti-MAG Antibodies

6.3.3. Patients With Anti-S-Carb Antibodies

7. Example: Characterization Of Neuroprotein-1

7.1. Protein Identification

7.2. Patient Testing

8. Example: Different Reactivity Of Serum IgM To GM1 Ganglioside AndCyclophilin A In Treatable Multifocal Motor Neuropathy

8.1. Materials And Methods

8.1.1. Patients

8.1.2. Antibody Assays

8.1.3. Protein Sequencing

8.1.4. Western Blot

8.2. Results

8.2.1. Western Blotting Of High Titer Anti-GM1 Sera

8.2.2. Characterization Of The 17 kD Protein

8.2.3. ELISA Measurement Of Serum IgM Reactivity To TheCyclophilin-A-Like Protein (CyPA)

8.3. Discussion

8.3.1. Fine Specificities Of Anti-GM1 Antibodies

8.3.2. Reactivity Of Anti-GM1 Sera With CyPA

8.3.3. Implications Of Anti-CyPA Reactivity In Anti-GM1 Sera

8.3.4. Diagnostic Testing

9. Example: Characterization Of Neuroprotein-3

9.1. Protein Identification

9.2. Patient Testing

10. Example: Neuroprotein-4

10. Example: Neuroprotein-5

12. Example: A Model of Disease Production By Inducing Anti-sulfatideAntibodies In Experimental Animals

13. Example: Monoclonal Antibodies To Neuroprotein-1

14. Example: Monoclonal Antibodies To Neuroprotein-2

15. Example: Monoclonal Antibodies To Neuroprotein-3

1. INTRODUCTION

The present invention relates to methods of diagnosing peripheralneuropathies that comprise determining the titer of antibodies directedtoward particular nervous system antigens. It also provides forsubstantially purified preparations of specific antigens namelyneuroprotein-1, neuroprotein-2, neuroprotein-3, neuroprotein-4 andneuroprotein-5, which may be used in such diagnostic methods.

2. BACKGROUND OF THE INVENTION 2.1. Peripheral Neuropathies

A patient who exhibits a disorder of one or more peripheral nerves issaid to suffer from a peripheral neuropathy. Peripheral nerves extendbeyond the brain and spinal cord into tissues that lie outside thecentral nervous system to provide a bidirectional communication network.They serve as conduits of impulses from the brain and spinal cord to therest of the body; for example, motor neurons carry signals to directmovement. Peripheral nerves are also capable of transmitting sensoryinformation gathered by specialized receptors to the brain. In short,peripheral nerves provide the connection between brain, body, andenvironment, and serve to coordinate the relationship between anorganism's brain and the outside world.

A peripheral neuropathy may manifest itself in a number of ways. If amotor nerve is affected, the patient may exhibit weakness in the musclegroups supplied by that nerve. If a sensory nerve is involved, thepatient may experience numbness, tingling, loss of sensitivity totemperature, touch, and/or vibration, or even increased sensitivity inthe area innervated by the diseased nerve.

Numerous varieties of peripheral neuropathy exist. Some are common,others are extremely rare. The etiology of certain peripheralneuropathies is well understood but some remain a mystery. Manyneuropathies have been classified into particular syndromes. Eachsyndrome is associated with its own set of clinical symptoms and signs,prognosis, and treatment options. It is extremely important to be ableto match a particular patient with the syndrome that corresponds to hisor her clinical condition. Such matching, like a road map, permits thephysician to choose a course of treatment and to counsel the patient asto prognosis. Often the identification of a syndrome alerts thephysician to another medical condition associated with the patient'speripheral neuropathy which requires a particular course of treatmentand carries its own prognosis. Accordingly, the ability to make acorrect and precise diagnosis is exceedingly important in the managementof a patient suffering from a peripheral neuropathy. Making the correctdiagnosis may, however, be difficult. In the past, such diagnosis hasdepended upon an analysis of the patient's symptoms and an extremelydetailed physical examination. To further complicate matters, manyperipheral neuropathy syndromes have not yet been fully characterized.

Peripheral neuropathies may appear as manifestations of a wide varietyof disease processes, including genetic, traumatic, metabolic, immune,and vascular disorders, as shown by Table I (see, for review, Plum andPosner, 1985, in "Pathophysiology--The Biological Principles ofDisease," Smith and Thier, eds , Second Edition, W. B. Saunders Co.,Philadelphia, Pa., pp. 1085-1090).

                  TABLE I                                                         ______________________________________                                        ANATOMIC CLASSIFICATION OF                                                    PERIPHERAL NEUROPATHY                                                         TWO OVERALL TYPES -                                                           1. SYMMETRICAL GENERALIZED                                                    2. FOCAL AND MULTIFOCAL                                                       ______________________________________                                        1.  Symmetrical Generalized                                                                        Neuropathies (Polyneuropathies)                              Distal Axonopathies                                                                            Toxic - many drugs, industrial                                                and environmental chemicals                                                   Metabolic - uremia, diabetes,                                                 porphyria, endocrine                                                          Deficiency - thiamine,                                                        pyridoxine                                                                    Genetic - HMSN II                                                             Malignancy associated - oat-cell                                              carcinoma, multiple myeloma                                  Myelinopathies   Toxic - diphtheria, buckthorn                                                 Immunologic - acute inflam-                                                   matory                                                                        polyneuropathy (Guillain-Barre)                                               chronic inflammatory                                                          polyneuropathy                                                                Genetic - Refsum disease,                                                     metachromatic leukodystrophy                                 Neuronopathies   Undetermined - amyotrophic                                   somatic motor    lateral sclerosis                                                             Genetic - hereditary motor                                                    neuronopathies                                               somatic sensory  Infectious - herpes zester                                                    neuronitis                                                                    Malignancy-associated - sensory                                               neuronopathy syndrome                                                         Toxic - pyridoxine sensory                                                    neuronopathy syndrome                                                         Undetermined - subacute sensory                                               neuronopathy syndrome                                        autonomic        Genetic - hereditary                                                          dysautonomia (HSN IV)                                    2.  Focal (Mononeuropathy) and Multifocal (Multiple                               Mononeuropathy) Neuropathies                                                  Ischemia - polyarteritis, diabetes, rheumatoid                                arthritis                                                                     Infiltration - leukemia, lymphoma, granuloma,                                 Schwannoma, amyloid                                                           Physical injuries - severance, focal crush,                                   compression, stretch and traction, entrapment                                 Immunologic brachial and lumbar plexopathy                                ______________________________________                                         From Schaumburg, H., Spencer, P., and Thomas, P. K.: Disorders of             Peripheral Nerves, Philadelphia, F. A. Davis Co., 1983.                  

Neuropathies may be classified on the basis of the anatomic component ofperipheral nerve most affected. For example, some peripheralneuropathies, such as Guillain-Barre syndrome, which is associated withinflammation of peripheral nerve, is classified as a demyelinatingneuropathy because it is associated with destruction of the myelinsheath that normally surrounds the nerve cell axon. In contrast, axonalneuropathies result from damage to the axon caused either by directinjury or, more commonly, from metabolic or toxic injury. In axonalneuropathy, the myelin sheaths disintegrate, as in demyelinatingneuropathy, but myelin loss is secondary to deterioration of the axon.Still other neuropathies, classified as neuronopathies, are caused bydegeneration of the nerve cell body; examples include amyotrophiclateral sclerosis and herpes zoster neuronitis.

Peripheral neuropathies are also classified according to thedistribution of affected nerves. For example, as shown in Table I, someneuropathies are symmetrically, generally distributed, whereas othersare localized to one or several areas of the body (the focal andmultifocal neuropathies).

Yet another characteristic used to categorize peripheral neuropathies isthe nature of the patient's symptoms, i.e., whether the patient sufferspredominantly from sensory or motor abnormalities. Some peripheralneuropathies, such as amyotrophic lateral sclerosis (ALS) and therecently described Multifocal Motor Neuropathy (MMN) with conductionblock are associated primarily with motor dysfunction. Others, such asparaneoplastic sensory neuropathy and neuronopathy associated withSjogren's syndrome, are manifested by sensory abnormalities.

A brief description of several disorders of peripheral nerves asfollows.

2.1.1. Amyotrophic Lateral Sclerosis

Of the predominantly anterior horn cell (AHC) disorders, amyotrophiclateral sclerosis (ALS or Lou Gehrig's disease) is the most common (seeWilliams and Windebank, 1991, Mayo Clin. Proc. 66:54-82 for review).

The initial complaint in most patients with ALS is weakness, morecommonly of the upper limbs (Gubbay et al., 1985, J. Neurol.232:295-300; Vejjajiva et al., 1967, J. Neurol. Sci. 4:299-314; Li etal., 1988, J. Neurol. Neurosurg. Psychiatry 51:778-784). Usually theearly pattern of weakness, atrophy, and other neurological signs isasymmetric and often focal (Munsat et al., 1988, Neurol. 38:409-413).Muscle cramps, paresthesias (tingling sensations) and pain are frequentcomplaints (Williams and Windebank, supra). Widespread fasciculationsare usually present (id.). The rate of progression of the disease variesfrom patient to patient (Gubbay et al., 1985, J. Neurol. 232:295-300),but in virtually all cases the disease eventually results in completeincapacity, widespread paralysis (including respiratory paralysis) anddeath.

Anatomically, the most prominent changes are atrophy of the spinal cordand associated ventral roots and firmness of the lateral columns (hencethe name, amyotrophic lateral sclerosis; Williams and Windebank, supra).Upper motor neurons are also involved and degenerate in ALS. The brainmay appear normal macroscopically, although atrophy of the motor andpremotor cortices is usually present due to upper motor neuroninvolvement. There is widespread loss of Betz cells and other pyramidalcells from the precentral cortex, with consequent reactive gliosis(Hammer et al., 1979, Exp. Neurol. 63:336-346).

Current treatment consists of symptomatic therapy to diminish musclecramps, pain, and fatiguability. Prosthetic devices are used tocompensate for muscle weakness. Pharmacologic therapy to alter theprogress of the disease has, however, been largely unsuccessful.Putative therapeutic benefits of thyrotropin releasing hormone have metwith conflicting results (Brooks, 1989, Ann. N.Y. Acad. Sci.553:431-461). Administration of gangliosides has been ineffective(Lacomblez et al., 1989, Neurol. 39:1635-1637). Plasmapheresis has shownno therapeutic advantage both alone and in combination withimmunosuppressive treatment (Olarte et al., 1980, Ann. Neurol.8:644-645; Kelemen et al., 1983, Arch. Neurol. 40:752-753). Theantiviral agent guanidine was reported to have potential short-termbenefits, but the results were not reproducible (Munsat et al., 1981,Neurol. 31:1054-1055). Administration of branched-chain amino acids toactivate glutamate dehydrogenase was reported to slow the rate ofdecline of patients in an abbreviated study (Plaitakis et al., 1988,Lancet :1:1015-1018). Most recent therapeutic trials, some in progress,involve whole-body total lymphoid irradiation, the use of amino acidsN-acetyl-cysteine, N-acetylmethionine, L-threonine, and long-termintrathecal infusion of thyrotropin releasing hormone (Williams andWindebank, supra).

Animal models that bear clinical and pathologic resemblances to ALSinclude the MND mouse, an autosomal dominant mutant exhibitinglate-onset progressive degeneration of both upper and lower motorneurons (Messer and Flaherty, 1986, J. Neurogen. 345-355); the wobblermouse, that exhibits forelimb weakness and atrophy in early life due tomuscle denervation, and hereditary canine spinal muscular atrophy in theBrittany spaniel (Sack et al., 1984, Ann. Neurol. 15:369-373; Silleviset al., 1989, J. Neurol. Sci. 91:231-258; Bird et al., 1971, ActaNeuropathol. 19:39-50).

2.1.2. Multifocal Motor Neuropathy with Conduction Block

In previous years, patients suffering from multifocal motor neuropathy(MMN) with conduction block were often considered to have pure motorforms of chronic inflammatory demyelinating polyneuropathy (CIDP) orlower motor neuron (LMN) forms of ALS (Bird, 1990, Current OpinionNeurol. Neurosurg. 3:704-707). MMN has recently been characterized as adistinct clinical syndrome. MMN appears to be characterized clinicallyby asymmetric, progressive, predominantly distal limb weakness; arms areinvolved more frequently than legs and there is generally no bulbar,upper motor neuron, or sensory involvement (Id.). In more than eightypercent of patients the weakness begins in the hands and may progressslowly for periods up to twenty years. MMN is more common in males thanfemales (2:1) and frequently (66 percent) begins in patients youngerthan 45 years of age. Nerve conduction studies show evidence ofmultifocal conduction block on motor but not sensory axons (Chad et al.,19.86, Neurology 36:1260-1266; Parry and Clarke, 1988 Muscle Nerve11:103-107; Pestronk et al., 1988, Ann. Neurol. 24:73-78).

Patients suffering from MMN appear not to improve clinically withcorticosteroid therapy; Pestronk et al. (1990, .Ann. Neurol. 27:316-326)noted improvement in only one out of seven patients treated withhigh-dosage prednisone; treatment with cyclophosphamide appeared to bemore successful. Pestronk et al. (1989, Neurology 39:628-633) havesuggested that prednisone and cyclophosphamide may exert differenteffects on autoantibodies in neuromuscular disorders.

MMN may be distinguishable from another motor neuropathy syndrome thatmore clearly meets criteria for a diagnosis of chronic inflammatorydemyelinating polyneuropathy (CIDP). Although both are predominantlymotor neuropathies, MMN and motor CIDP differ in their clinicalfeatures, physiologic changes, serologic findings and response toimmunosuppression. In contrast to MMN, patients with motor CIDP usuallyhave symmetric weakness that involves proximal muscles early in thecourse of the disease.. While nerve conduction studies in CIDP may showevidence of conduction block, there is often evidence of more diffusedemyelination on both motor and sensory axons. Physiologic changes inmotor CIDP that are found in only a minority of patients with MMNinclude slowing (less than 70% of normal) of conduction velocities, andprolonged distal latencies to the range found in demyelinatingdisorders. High titers of IgM anti-GM1 antibodies are only rarely foundin motor CIDP patients. A further contrast to MMN is the response totreatment. As has been reported for the overall population of CIDPpatients, those with motor CIDP often demonstrate increased strengthwithin a few weeks to months after treatment with prednisone,plasmapheresis or intravenous human immune globulin.

2.1.3. Sensory Neuropathies

A variety of neuropathies are primarily sensory in nature, includingleprous neuritis, sensory perineuritis, hyperlipidemic neuropathies,certain amyloid polyneuropathies, and distal symmetrical primary sensorydiabetic neuropathy. These are primary axonal or demyelinatingneuropathies.

In addition, pure sensory syndromes, known as sensory neuronopathies,have been identified that result from primary pathological events in thedorsal root ganglion or trigeminal cell bodies (Asbury and Brown, 1990,Current Opinion. Neurol. Neurosurg. 3:708-711; Asbury, 1987, Semin.Neurol. 7:58-66). Some examples of sensory syndromes follow.

A severe subacute primary sensory neuropathic disorder may occur in thecontext of concurrent malignancy, particularly small-cell lung cancer,and may in fact precede the diagnosis of malignancy (Asbury and Brown,supra).

Sjogren's syndrome, characterized by dry mucous membranes and skin andthe destruction of salivary and lacrimal glands, appears to beassociated with a sensory neuronopathy. Griffin et al. (1990, Ann.Neurol. 27:304-315) found that eleven women and two men with undiagnosedataxic sensory neuronopathy and autonomic dysfunction all had primarySjogren's syndrome.

Furthermore, hundreds of commonly encountered chemical[s, includingenvironmental toxins, vitamins, and various prescription drugs, cancause a polyneuropathy that begins as a distal symmetrical sensoryneuropathy and may progress to a mixed sensory-motor-autonomic disorder.Examples of such chemicals include cis-platinum (Mollman, 1990, N. Engl.J. Med. 322:126-127), vitamin B₆ (Xu et al., 1989, Neurology39:1077-1083), taxol (Lipton et al., 1989, Neurology 39:368-373) anddoxorubicin (in experimental animals) (Asbury and Brown, supra).

However, the majority of predominantly sensory neuropathies in patientsremain undiagnosed.

2.2. Antigenic Structures of the Peripheral Nervous System

There is increasing evidence that serum antibodies directed againstglycolipids or glycoproteins (Table II) commonly occur in high titer inpatients with some forms of motor neuron disease and peripheralneuropathy. This association was first noted in patients with chronicdemyelinating neuropathies who had monoclonal IgM serum antibodies thatreacted with myelin-associated glycoprotein. It is now apparent thathigh titers of serum antibodies to GM1 ganglioside commonly occur alongwith lower motor neuron (LMN) diseases and motor neuropathies.Antineuronal antibodies in serum and CSF have been identified inpatients with sensory ganglionopathies and small-cell lung neoplasms. Wewill review the association of clinical neuromuscular syndromes withantibodies that react with glycolipids and structurally relatedglycoproteins.

                  TABLE II                                                        ______________________________________                                        COMMON ANTIGENIC TARGETS IN                                                   NEUROPATHY SYNDROME PATIENTS                                                  Compound                                                                              Structure                                                             ______________________________________                                        GM1     Galβ1-3Ga1NAcβ1-4Ga1β1-4G1cβ1-1'Ceramide                  3                                                                             Neu5Acα2                                                        GA1     Galβ1-3Ga1NAcβ1-4Ga1β1-4G1cβ1-1'Ceramide          GM2     Ga1NAcβ1-4Galβ1-4G1cβ1-1'Ceramide                              3                                                                             Neu5Acα2                                                        Sulfatide                                                                             SO.sub.4 -3-Galβ1-1'Ceramide                                     MAG and So.sub.4 -3-Glucuronic Acid -                                         SGPG    antigenic epitope                                                     ______________________________________                                         Common antigenic targets in neuropathy syndrome patients. Structures of       GM1 ganglioside, asialoGM1 ganglioside (GA1), and GM2 ganglioside are         illustrated. The gangliosides GM1 and GM2 consist of a) a lipid component     ceramide, b) a carbohydrate moiety (3 sugars for GM2, 4 sugars for GM1)       that includes galactose (Gal), galactosamine (GalNAc) and glucose (Glc),      and c) a sialic acid ganglioside. GD1a has an additional sialic acid          attached to the terminal galactose on GM1. GD1b has a second sialic acid      attached to the sialic acid on GM1. GT1b has additional sialic acid in        both locations.                                                          

2.2.1. Gangliosides

Gangliosides are a family of acidic glycolipids that are composed oflipid and carbohydrate moieties (Table II). The lipid moiety, ceramide,is a fatty acid linked to a long chain base, sphingosine. In mammalianbrain gangliosides the sphingosine contains 18-20 carbon atoms. Thecarbohydrate portion of gangliosides is a series of 2 or more sugarswith at least one sialic acid. The major gangliosides in mammalian braincontain 1-3 sialic acids, usually N-acetylneuraminic acid (Neu5Ac), anda chain of 2-4 other sugars. Four gangliosides are especially abundantin brain, namely GM1, GD1a, GD1b and GT1b. They each contain the same 4sugar chain (Table II) but vary in the number of sialic .acid molecules;GM1 with one, GD1a and GD1b with two and GT1b with three. In peripheralnerve a fifth ganglioside, LM1, containing a different carbohydratestructure, also occurs in relative abundance. Numerous minorgangliosides in brain, nerve and myelin have been described.Gangliosides generally reside in the outer layer of plasma membrane. Thehydrophilic sugars are located on the outer surface of the membrane.They are linked to the cell by the hydrophobic lipid moiety which isinserted into the membrane.

GM1 is one of the most abundant gangliosides in neuronal membranes butis unusual outside of the nervous system. It has been postulated thatgangliosides may play a role in membrane and cell functions. There is alarge literature suggesting that administration of exogenous GM1ganglioside enhances neurite outgrowth and recovery from injury. GM1 andother gangliosides can function as cellular receptors. The binding ofcholera toxin to GM1 ganglioside is well documented. Gangliosides onnerve terminals may also serve as receptors for tetanus and botulinumtoxins.

The abundance of gangliosides in the nervous system and theextracellular location of their sugars suggests that they could beantigenic targets in autoimmune neurological disorders. The terminaldisaccharide on GM1, Galβ1-3GalNAc, is known to be antigenic when itoccurs on systemic glycoproteins. However, the disaccharide on theseglycoprotens is normally hidden from immune attack by a sialic acidattached to each sugar. Several investigators have tested sera frompresumed autoimmune disorders for antibody binding to panels ofgangliosides looking for possible targets of the immune processes.

2.2.2. Myelin-Associated Glycoprotein

Myelin-associated glycoprotein (MAG; Table II) is a nervoussystem-specific protein that is found in both the central and peripheralnervous systems. It is present in myelin related membranes but not thecompact myelin of oligodendrocytes and Schwann cells. MAG is an integralmembrane protein. Almost one third of its molecular weight is due to thepost-translational addition of carbohydrate molecules. The terminalsulfated glucuronic acid carbohydrate moieties in MAG are importantbecause they are the main targets of IgM paraprotein antibodyreactivity. MAG has structural similarities to immunoglobulins and tocell adhesion molecules. MAG is thought to mediate adhesive and trophicinteractions between cell membranes during myelin formation andmaintenance. Sulfated glucuronic acid epitopes also occur on peripheralnerve glycolipids including sulfated glucuronal paragloboside (SGPG) anda group of glycoproteins of molecular weight 19,000 to 28,000.

2.3. Antibodies in Peripheral Neuropathies

There has been a growing appreciation that many neurologic disorders mayhave an autoimmune basis. This realization has occurred in conjunctionwith an increasing knowledge of the molecular specificities ofautoantibodies (Steck, 1990, Neurology 40:1489-1492). Consequently, therole of antibody testing as part of the neurologic diagnostic processhas become progressively more important.

2.3.1. Anti-GM₁ Antibodies

Pestronk et al. (1990, Ann. Neurol. 27:316-326) reports a study of serafrom 74 patients with lower motor neuron syndromes. Antibodyspecificities were compared to clinical and electrophysiological data inthe same patients. Several distinct lower motor neuron syndromes wereidentified based on clinical, physiological, and antiglycolipid antibodycharacteristics. The results indicated that antibodies to gangliosideGM₁, to similar glycolipids, and to carbohydrate epitopes on GM₁ and GA₁may be common in sera of patients with lower motor neuron syndromes.

Similarly, Nobile-Orazio et al. (1990, Neurology 40:1747-1750) reports astudy that compared anti-GM₁ IgM antibody titers by enzyme-linkedimmunosorbent assay in 56 patients with motor neuron disease, 69patients with neuropathy, and in 107 control subjects. Anti-GM₁ IgMantibodies were found in 13 (23 percent) of motor neuron diseasepatients, 13 ( 18.8 percent) neuropathy patients, and 8 (7 percent) ofcontrols. Two of the 13 neuropathy patients exhibiting anti-GM₁ antibodyalso were found to have antibodies directed toward MAG protein.

It appears that high titers of serum IgM anti-GM1 ganglioside antibodies(present at dilutions of >350-400) occur commonly in some motor neuronand peripheral neuropathy syndromes but not in others (Table III). Thehighest titers (>7,000) are especially specific for lower motor neuronsyndromes and multifocal motor neuropathy. Low titers of anti-GM1antibodies (<350) are not specific. They may be found in sera frompatients with a variety of neurologic and autoimmune disorders as wellas from some normal controls.

TABLE III IgM ANTI-GM₁ ANTIBODIES--CLINICAL ASSOCIATIONS

1) Frequently (>50%) present in high titer (>350):

Multifocal motor neuropathy

Distal lower motor neuron syndromes

2) Occasionally (5-15%) present in high titer:

Proximal lower motor neuron syndromes

ALS

Guillain-Barre Syndrome

Polyneuropathies=especially motor-sensory & asymmetric

Autoimmune disorders without neuropathy

3) Rarely (<5%) present in high titer:

CIDP

Sensory neuropathies & neuronopathies

Normals (<1% )

2.3.2. Anti-Mag Antibodies

High titers of serum antibodies directed against MAG are commonlyassociated with a slowly progressive demyelinating peripheralneuropathy. In 40-50% of patients with IgM monoclonal gammopathy andneuropathy the M-protein reacts with MAG. The clinical syndrome relatedto high titers of serum anti-MAG antibodies is a distal symmetricneuropathy involving both sensory and motor modalities. Symptoms usuallybegin distally and symmetrically in the feet and legs. The hands arecommonly also affected. Unlike another demyelinating neuropathy, CIDP,weakness only involves proximal musculature late in the disorder.Sensory findings usually include large fiber dysfunction, with sensoryataxia in severe cases. The neuropathy is slowly progressive and mayapparently stabilize for long periods at a point of severe, or onlymild, dysfunction. A majority of patients with IgM anti-MAG relatedpolyneuropathy are male (>80%). Most are older than 50 years of age.Electrophysiological studies usually are indicative of demyelination.The most consistent finding is prolonged distal latencies. Conductionvelocity slowing, temporal dispersion and increased F-response latencyare also seen. Cerebral spinal fluid (CSF) protein concentration isoften elevated. Sera with very high titers of IgM anti-MAG activity showevidence of a monoclonal IgM in many cases if sensitive screeningmethods, such as immunofixation, are used. In contrast, patients withpredominantly sensory neuropathies, or those that are primarily axonal,only rarely have high-titer serum IgM reactivity to MAG (Nobile-Orazio,et al., 1989, Ann. Neurol. 26:543-550; Dubas et al., 1987, Cas. Rev.Neurol. (Paris) 143:670-683.

A common feature of the anti-MAG antibodies in demyelinating sensorymotor neuropathy syndromes is cross reactivity with compounds that, likeMAG, contain sulfate-3-glucuronate epitopes. These compounds includemyelin components such as the P_(o) glycoprotein (Bollensen et al.,1988, Neurology 38: 1266-1270; Hosokawa et al., 1988, In"Neuroimmunological Diseases," A Igata, ed Tokyo: University of TokyoPress, pp. 55-58) and an acidic glycolipid, sulfate-3-glucuronylparagloboside (SGPG) 5(Nobile-Orazio, supra; Ilyas et al., 1985, Proc.Natl. Acad. Sci. U.S.A. 82:6697-6700; Chou et al., 1986, J. Biol. Chem.261: 11717-11725; Ariga et al., 1987, J. Biol. Chem. 262: 848-853).

3. SUMMARY OF THE INVENTION

The present invention relates to methods of diagnosing peripheralneuropathies that comprise determining the titer of antibodies directedtoward specific nervous system antigens. It is based on the discoverythat the presence of elevated titers of certain antibodies correlateswith particular clinical and anatomical characteristics.

The present invention, in part, relates to diagnostic methods whichdetermine the presence of antibodies directed toward antigens thatcomprise a SO₄ -3-galactose moiety, including sulfatide antigen. In apreferred embodiment of the invention, the presence of high titers ofanti-sulfatide antibodies in a patient's serum supports a diagnosis of apredominantly sensory axonal neuropathy.

The invention is also based on the discovery and characterization of anumber of nervous system proteins, including neuroprotein-1,neuroprotein-2, neuroprotein-3, neuroprotein-4, and neuroprotein-5. Eachof these proteins is recognized by antibodies in patients suffering fromperipheral neuropathies, and therefore may be used in diagnostic methodsto identify and define particular neuropathic syndromes.

The correlation between elevated antibody titers toward specificantigens and the clinical and anatomical features of peripheralneuropathies provides an objective standard[of diagnosis and allows forthe categorization of patients into groups that share similar prognosesand treatment options. The detection of elevated titers of particularantibodies may serve as an early marker of neurologic disease, and maypermit treatment of the patient's condition before irreversible damagehas occurred. In addition, the characterization of antigen/antibodypairs according to the invention may serve as valuable tools in thestudy of the genesis of peripheral neuropathies.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Western blot of human sera versus myelin proteins (seradilutions=1:1000). Lane 1 illustrates that sera with high ELISA anti-MAGactivity (e.g. patient No. 20) stain MAG on Western blot. Lane 2illustrates that selective antisulfatide sera by ELISA (e.g. patient No.3) do not stain MAG. Normal sera at 1:1000 dilution also do not stainMAG.

FIG. 2. A. Orcinol staining of HPTLC of standards. Lane 1=sulfatidedoublet; Lane 2=bovine brain gangliosides. B. Immunostaining of HPTLCseparation of mixture of gangliosides and sulfatide (seradilutions=1:1000). Lane 1 illustrates that antisulfatide sera (e.g.patient No. 3) stain the sulfatide doublet but not gangliosides. Normalcontrols and sera that react selectively with MAG using ELISAmethodology produce no staining.

FIG. 3. Amino acid sequence of amino terminus of neuroprotein-2 (SEQ. IDNO: 1) and comparison to Cyclophilin A (SEQ ID NO: 2).

FIG. 4. Amino acid (SEQ. ID NO: 3) sequence of amino terminus ofneuroprotein-3 compared with the amino acid sequence of human β-tubulin(SEQ ID NO: 4)

FIG. 5. Western blot of serum (W1160; 1:1000) versus neural proteins.Lanes 2 to 4 illustrate reactivity of serum W1160 with NP-1 bands withmolecular weights of approximately 36 kD, 38 kD, and 42 kD. Molecularweight standards above and below NP-1 are indicated in lane 1.

FIG. 6. Western blot of serum IgM (dilution=1:1000) versus thenon-myelin fraction of human CNS (100 μg protein per lane). Lanes 1 to 5show binding of individual anti-GM1 sera from patients with treatableMMN. Lanes 6 to 10 show binding of ALS anti-GM1 sera. Lanes 11 to 15show binding of PN anti-GM1 sera. Lane 16 shows binding of a pooledcontrol serum. Note that MMN sera do not bind well to the 17 kD band(arrow). ALS sera bind to this band but only unusually to others. PNsera bind to the 17 kD band and others with higher or lower molecularweight as well.

FIG. 7. IgM antibody titers versus GM1 ganglioside in patients with MMNresponsive to treatment (MMN Rx), other cases of MMN, LMN syndromes, ALSand peripheral neuropathies (PN). Sera were selected for anti-GM1antibody titers>350. Note that there is considerable overlap betweenpatient groups. The highest titers (>7000) were more common in MMN andLMN than in ALS or PN groups.

FIG. 8. IgM antibody titers versus CyPA in sera with high titers (>350)of IgM anti-GM1 antibodies. Diagnostic groups are the same as in FIG. 7.Very low titers (<300) were more common in MMN than in the otherdiagnostic group. Very high titers (>7000) only occurred in LMN, ALS andPN groups.

FIG. 9. Values of ratios of IGM antibody titers to CyPA compared to GM1(CyPA:GM1 antibody ratio) in individual sera. Most MMN sera (82%) haveratios <0.79. Most ALS and PN sera (90%) have ratios >0.79.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to autoantibodies and their targets in thediagnosis of peripheral neuropathies. For purposes of clarity, and notby way of limitation, the detailed description of the invention isdivided into the following subsections:

(i) characterization of antigens;

(ii) methods of diagnosis of peripheral neuropathies;

(iii) preparation of antibodies; and

(iv) additional utilities of the invention.

5.1. Characterization of Antigens

The present invention relates to a number of antigens. In variousembodiments, it provides for antigens that comprise at least one SO₄-3-galactose moiety, including, but not limited to, sulfatide. Inadditional embodiments, the present invention provides for substantiallypurified neuroprotein-1 (NP-1), neuroprotein-2 (NP-2) , neuroprotein-3(NP-3) , neuroprotein-4 (NP-4) and neuroprotein-5 (NP-5) , describedinfra.

The present invention ]provides for substantially purified NP-1, asexemplified in Section 7, infra. Substantially purified NP-1 accordingto the invention consists essentially of three related protein moleculeshaving a molecular weight of about 36, 38 and 42 kD. NP-1 is expressedat higher levels in central nervous system (CNS) and spinal cordnon-myelin white matter compared to other tissues. It may be prepared,for example, and not by limitation, by processing white matter tissueobtained from human brain by homogenizing white matter in 0.88M sucroseand then centrifuging the lysate in a discontinuous sucrose gradientwith layers of 0.32M and 0.88M sucrose for 30 minutes at 34,000 rpm. Theresulting pellet may then be purified by delipidation in a mixture ofether and ethanol at a ratio of about 3:2 for 10 minutes at roomtemperature and then washing the pellet three times in 1% Triton-X-100.After delipidation and after the first two washes each pellet may berecovered by centrifuging at 10,000 rpm for 10-20 minutes. After thethird wash the pellet may be recovered by centrifuging at 20,000 rpm for20 minutes. NP-1 in the pellet may then be taken into solution in 0.1MTris, 0.2 mM PMSF and 0.5 mM EDTA at pH 7.2 and subjected topolyacrylamide gel electrophoresis (PAGE) in a 12 percent polyacrylamidegel to separate its components. Three protein bands having apparentmolecular weights of about 36, 38 and 42 kD may then be identified andseparated from the rest of the gel, for example, by cutting out slicesof the gel that correspond to those bands. The NP-1 protein in the bandsmay then be eluted into suitable buffer using standard techniques.

The present invention further provides for a substantially purifiedneuroprotein-1 having a molecular weight of about 36 kD, a substantiallypurified neuroprotein-1 having a molecular weight of about 38 kD, and asubstantially purified neuroprotein-1 having a molecular weight of about42 kD.

The present invention also provides for substantially purified NP-2, asexemplified in Section 8, infra. Substantially purified NP-2 accordingto the invention consists essentially of two related protein moleculeshaving a molecular weight of about 17-18 kD. NP-2 is identifiable as twobands that migrate immediately below the large myelin basic protein bandon 15% Coomassie blue-stained PAGE of CNS white matter.

NP-2 is expressed at higher levels in CNS white matter and peripheralnerve compared to other tissues. NP-2 comprises the amino acid sequencesubstantially as set forth in FIG. 3, or a functionally equivalentsequence. As used herein, the term "functionally equivalent sequence" isconstrued to mean a sequence in which functionally equivalent amino acidresidues are substituted for residues within the sequence resulting in asilent change. For example, one or more amino acid residues within thesequence can be substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine; neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Based upon its amino acid sequence, NP-2 appears to bearhomology toward cyclophilin A.

NP-2 may be prepared, for example, and not by limitation, by processingwhite matter to produce a nonmyelin pellet as set forth supra for NP-1.The pellet may then be purified first by washing in deionized waterfollowed by centrifugation at 10,000 rpm for 20 minutes and then bydelipidation in a mixture of ether and ethanol at a ratio of about 3:2for 10 minutes at room temperature and then washing in 1% Triton-X-100.After delipidation and after each wash the pellet may be collected bycentrifugation at about 10,000 rpm for 10-20 minutes. The pellet maythen be washed (x3) in Tris buffer pH 7.2 containing 0.1MM PMSF and 0.5mM EGTA, then collected by centrifugation at 10,000 rpm for 20 minutes.The protein may then be dissolved from the pellet in a solution of 25mMChaps, 2M sodium chloride, lmM EGTA, 0.15M sodium phosphate, 2% glyceroland PMSF by incubation overnight at 4° C. Afterward, the dissolvedprotein may be collected by centrifuging the product of overnightincubation at 100,000 g for 2 hours and then recovering the supernatant.The supernatant may then be desalted and concentrated bymicroultrafiltration (e.g. AMICON) with a 1000 kD filter to form aconcentrate that may be subjected to PAGE, for example preparative PAGEusing a 15% polyacrylamide gel, to separate its components. Two proteinbands having apparent molecular weights of about 17 to 18kD may then beidentified and separated from the rest of the gel, for example, bycutting out slices of the gel that correspond to those bands. The NP-2protein in the bands may then be eluted into suitable buffer usingstandard techniques.

The present invention further provides for substantially purified NP-3,as exemplified in Section 9, infra. Substantially purified NP-3according to the invention has a molecular weight of 50-54KD. Itcomprises an amino terminal amino acid sequence substantially as setforth in FIG. 4, (SEQ. ID NO: 3) which shows strong homology tobeta-tubulin, and is immunologically cross-reactive with beta tubulin.On 12% PAGE analysis it migrates just above the location of Wolfgramproteins in a separation of human white matter or myelin. It may beprepared, for example, and not by limitation, from myelin harvested fromhuman brain according to the method of Norton and Poduslo, 1973, J.Neurochem, 21: 1171-1191. The CNS myelin proteins may be purified bydelipidation using a mixture of ether and ethanol at a ratio of 3:2 andthen washing first with 1% Triton-X-100 three times. Pellets after eachof these washes may be obtained by centrifuging at 10,000 rpm for 10-20minutes. The protein in the final pellet so obtained may then bedissolved in 2 percent SDS and then subjected to PAGE on a 12%polyacrylamide gel. A protein band having an apparent molecular weightof about 50 to 54 kD may then be identified and separated from the restof the gel, for example, by cutting out slices of the gel thatcorrespond to those bands. The NP-3 protein in the bands may then beeluted into suitable buffer using standard techniques.

The present invention also provides for substantially purified NP-4, asexemplified in Section 10, infra. Substantially purified NP-4 has amolecular weight of about. 20 to 24 kD. NP-4 may be prepared, forexample, and not by limitation by the method as set forth for NP-2(supra), and including the washing (x3) in Tris buffer. The pellet isthen dissolved in 2 percent SDS and subjected to PAGE on a 15 percentpolyacrylamide gel. A protein band having an apparent molecular weightof about 20-24 kD may then be identified and separated from the rest ofthe gel by the methods outlined for NP-2 and NP-3.

The present invention also provides for substantially purified NP-5, asexemplified in Section 11, infra. Substantially purified NP-5 has amolecular weight of about. 30-32 kD. It may be prepared for example, andnot by limitation, by differential centrifugation washing and elution ofspecific 30-32 kD bands from PAGE gels using methods similar to thosedescribed in NP-4.

The present invention provides for substantially purified NP-1, NP-2,NP-3, NP-4, and NP-5 having the characteristics of a protein that isprepared by a process exemplified, respectively, in Sections 7, 8, 9,10, and 11, infra, and briefly described above. However, the presentinvention also provides for NP-1, NP-2, NP-3, NP-4, and NP-5 that areprepared by different methods, including different purificationstrategies, chemical synthesis, and recombinant DNA technology, etc.,provided that the characteristics exhibited by the respective proteinamong Example Sections 7 through 11 are substantially retained.

The present invention further provides for fragments and derivatives ofNP-1, NP-2, NP-3, NP-4, and NP-5. Fragments are construed to be at leastsix amino acids in length. Derivatives include the products ofglycosylation, deglycosylation, phosphorylation, reduction, oxidation,or conjugation of the proteins of the invention to another protein ornon-protein molecule. In preferred, nonlimiting embodiments of theinvention the fragment or derivative is immunogenic.

The present invention further provides for a substantially purifiedprotein having a molecular weight of about 22 kD that binds tomonoclonal antibody B3H12.

The present invention further provides for a substantially purifiedprotein having a molecular weight of about 10-12 kD that binds to B5G12.

The present invention further provides for a substantially purifiedprotein having a molecular weight of about 22 kD that binds to B5G12.

The present invention also provides for a substantially purified proteinhaving a molecular weight of about 34-38 kD that binds to B5G10.

The present invention still further provides for a substantiallypurified protein having a molecular weight of about 55-65 kD that bindsto B5G10.

The antibodies of the invention, in particular A1A1.6, A2H3.7, A2H10.1,A5H10.1, B3H12, B5G10, B5G12, B5H10, C1F10, C2F3, C1H3, and C2H1, may beused to prepare substantially pure preparations of their target antigensby immunoprecipitation or affinity chromatography.

5.2. Methods of Diagnosis of Peripheral Neuropathies

The present invention provides for methods of diagnosing peripheralneuropathies based upon determining the titer of antibody directedtoward SO₄ -3-galactose, sulfatide, cyclophilin, tubulin (preferably,β-tubulin), NP-1, NP-2, NP-3, NP-4 or NP-5 antigen.

According to the invention, a peripheral neuropathy may be diagnosed ina patient by determining that the titer of antibodies in a patientsample directed toward SO₄ -3-galactose, sulfatide, cyclophilin, tubulin(preferably, β-tubulin), NP-1, NP-2, NP-3, NP-4 or NP-5 is greater thanthe number of antibodies which may be present in a comparable samplefrom a normal blood, serum, cerebrospinal fluid, nerve tissue, braintissue, urine, nasal secretions, saliva, or any other body fluid ortissue. Although the following embodiments relate to the determinationof antibody titers in serum, these represent preferred but nonlimitingembodiments of the invention, which may be analogously applied to anypatient sample as described above.

In particular embodiments, the present invention provides for a methodof diagnosing a peripheral neuropathy in a patient comprisingdetermining the titer of antibody that binds to an antigen comprising atleast one SO₄ -3-galactose moiety in a serum sample from the patient, inwhich a high titer correlates positively with a predominantly axonalneuropathy. In preferred embodiments, this neuropathy is predominantlysensory in nature. A high titer of IgG antibody is construed to begreater than about 1:900; if the antibody is IgM, then a high titer isconstrued to be greater than 1:1100. In particularly preferredembodiments of the invention, the antigen comprising SO₄ -3-galactose issulfatide, and the predominantly axonal, predominantly sensoryneuropathy has a clinical history of presenting first as numbness andparesthesias or pain in the feet, and then spreading more proximally inthe legs and eventually involving first the hands and then the arms.Mild weakness may be noted in some patients, but may not begin forseveral months or years after the onset of sensory complaints. Onexamination, sensory and motor signs may be more prominent distally.Reflexes may be diminished or absent at the ankles but are usuallypreserved elsewhere.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to cyclophilin in a serum sample from the patient.In patients with treatable MMN, the ratio of antibody titers tocyclophilin compared to GM1 ganglioside may be less than 0.79. Inpatients with other peripheral neuropathies and ALS this ratio may begreater than 0.79. The difference in ratio may be used to distinguishthe treatable MMN from the essentially untreatable ALS.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to tubulin in a serum sample from the patient, inwhich a titer greater than about 1:1000 correlates positively with an5inflammatory demyelinating polyneuropathy such as Guillain-Barresyndrome or chronic inflammatory demyelinating polyneuropathy.

The present invention also provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to neuroprotein-1 in a serum sample from the patientin which a titer greater than or equal to about 1:1000 correlatespositively with a mixed axonal and demyelinating sensory-motorpolyneuropathy (see Section 7, infra).

The present invention also provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to NP-2 in a serum sample from a patient, in which atiter greater than 1:1000 and preferably greater than 1:2000 combinedwith the presence of anti-sulfatide antibodies correlates positivelywith predominantly sensory or sensory motor signs and axonal ordemyelinating neuropathies. Further, the presence of high titers ofantibodies that are cross-reactive with GM1 and sulfatide correlatespositively with a diagnosis of motor neuron disease; "high titer" inthis case should be construed to mean a value of 1:1000 for either IgMand IgG antibody (See Section 8, infra). In preferred embodiments, thepresence of low titer antibody toward NP-2 and high titer antibodytoward GM1, or a ratio of NP-2:GM1 of less than 0.79 supports adiagnosis of MMN. The presence of high titers of antibody to NP-2 and toGM1, or a ratio of titers of NP-2:GM1 antibodies of greater than orequal to 0.79 supports a diagnosis of ALS or peripheral neuropathy.

The present invention further provides for a method of diagnosing aperipheral neuropathy in a patient comprising determining the titer ofantibody that binds to NP-3 in a serum sample from the patient, in whicha titer greater than about 1:1000 correlates positively with an5inflammatory demyelinating polyneuropathy. In specific, non-limitingembodiments the inflammatory demyelinating polyneuropathy isGuillain-Barre syndrome or chronic inflammatory demyelinatingpolyneuropathy (CIDP). As stated in Section 9, infra, antibodiesdirected toward NP-3 have been observed in high titer at the onset ofGuillain-Barre syndrome which decrease over the course of the disease.Accordingly, the presence of high titers of anti-NP-3 antibodies may bean early marker of Guillain-Barre Syndrome. High titers of anti-NP-3antibodies are present in 40-45 percent of patients with CIDP.

The present invention still further provides for a method of diagnosinga peripheral neuropathy in a patient comprising determining the titer ofantibody that binds to NP-4 in a serum sample from the patient in whicha titer greater than or equal to about 1:500 correlates positively witha peripheral neuropathy such as, for example, but not by limitation,Guillain-Barre Syndrome or chronic inflammatory demyelinatingpolyneuropathy.

According to the present invention, antibody titer may be determined byany method known to the art using standard techniques, including, butnot limited to, enzyme-linked immunosorbent assay (ELISA) and othersolid phase immunoassays, radioimmunoassay, nephelometry, rocketelectrophoresis, immunofluorescence, Western blot (immunoblot), etc. Ina specific,, non-limiting embodiment of the invention, antibody titermay be determined as exemplified in the specific case set forth fordetermining titers to antibodies to glycolipids and MAG in Section6.1.2., infra.

The present invention further provides for diagnostic kits to be usedaccording to the invention. Such kits mail comprise (i) substantiallypurified antigen, such as an antigen comprising a SO₄ -3-galactosemoiety, 5sulfatide, cyclophilin, tubulin, NP-1, NP-2, NP-3, NP-4, orNP-5; (ii) detectably labelled antibody "detector antibody" that bindsto human antibody. The detector antibody may comprise an antibody boundto a detectable compound, including, but not limited to, an enzyme,radioactive molecule, or fluorescent compound. In preferred embodimentsof the invention, the detector antibody may be bound to an enzyme thatmay react with an added substrate to yield a colored product; in suchembodiments the kit may preferably include a supply of the substrate. Inan especially preferred embodiment of the invention, the detectorantibody may be conjugated to horseradish peroxidase. Detector antibodymay be specific for a particular class of human antibody, for example,it may bind to human IgM, IgG, IgA, IgE, or IgD, preferably to theconstant region of the molecules. To use the kit, the antigen providedmay be adhered to a solid support and then exposed to serum collectedfrom a patient. The amount of patient antibody bound may then bedetermined using detector antibody. Titers of antibodies may then becalculated from the amount of detector antibody bound using standardconversion algorithms. For example, if detector antibody compriseshorseradish peroxidase, titers of antibody may be calculated as setforth in Pestronk et al. (1990, Ann. Neurol. 2.7:316-326).

5.3. Preparation of Antibodies

According to the invention, SO₄ -3-galactose containing antigen,sulfatide, cyclophilin, tubulin, NP-1, NP-2, NP-3 NP-4 or NP-5 orfragments or derivatives thereof, may be used as immunogen to generateantibodies.

To improve the likelihood of producing an immune response, the aminoacid sequence of a neuroprotein antigen may be analyzed in order toidentify portions of the molecule which may be associated with increasedimmunogenicity. For example, the amino acid sequence may be subjected tocomputer analysis to identify surface epitopes. Alternatively, thededuced amino acid sequences of a neuroprotein antigen from differentspecies could be compared, and relatively non-homologous regionsidentified; these non-homologous regions would be more likely to beimmunogenic across various species.

For preparation of monoclonal antibodies directed toward the antigens ofthe invention, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (1975 Nature 256:495-497), as well as the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today4:72), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, in "Monoclonal Antibodies and CancerTherapy," Alan R. Liss, Inc. pp. 77-96) and the like are within thescope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982,Meth.Enzymol. 92:3-16). Chimeric antibody molecules may be preparedcontaining a mouse (or other species) antigen-binding domain with humanconstant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.81:6851,Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of the antigens of the invention. Forthe production of antibody, various host animals can be immunized bySinjection with antigen, or fragment or derivative thereof, includingbut not limited to rabbits, mice, rats, etc. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and, Corynebacterium parvum.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe 2 Fab or Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

As exemplified in Sections 13, 14, and 15, a number of monoclonalantibodies directed toward antigens of the invention have been produced,including monoclonal antibodies A1A1.6, A2H3.7,A2H10.1, and A5H10.1 toNP-1; B3H12, B5G10, B5G12, and B5H10 to NP-2; and C1F10, C2F3, C1H3, andC2H1 to NP-3. The present invention provides for these antibodies andhybridomas that produce these antibodies. and their functionalequivalents. Functional equivalents of a monoclonal antibody areconstrued herein to refer to antibodies or antibody fragments thatcompetitively inhibit the binding of a monoclonal antibody to its targetantigen. The present invention also provides for fragments andderivatives of the antibodies of the invention.

Antibody produced by these methods may be used to bind to the antigensof the invention in vitro or in vivo. The use of such antibodies mayreveal aberrancies in the distribution or level of expression of theantigens of the invention; for example, peripheral nerve may be found tobe depleted of a particular antigen or may exhibit an overabundance ofan antigen in various peripheral neuropathies. Accordingly, theantibodies of the invention may be used in the diagnosis of peripheralneuropathies. For example, such antibodies may be applied to a samplewhich is a section of peripheral nerve or other tissue or fluid obtainedfrom a patient; if the level of antibody binding to antigen in thesample from the patient differs from the level of binding to acomparable sample from a normal, healthy person, the patient may sufferfrom a peripheral neuropathy or related condition.

The antibodies of the invention may also be used as antigens themselvesto produce anti-idiotype antibody that may be useful in the treatment ofcertain peripheral neuropathies.

The antibodies of the invention may be administered to a non-humananimal in order to produce a model system that may be used to study aperipheral neuropathy.

5.4. Additional Utilities of the Invention

In additional embodiments, the present invention may be used to createnon-human animal model systems for peripheral neuropathy and may be usedtoward the cloning and recombinant expression of the neuroproteinantigens of the invention.

In order to create non-human animal model systems for peripheralneuropathy, an antigen of the invention, such as an antigen thatcomprises at least one SO₄ -3-galactose moiety, sulfatide, cyclophilin,tubulin, NP-1, NP-2, NP-3, NP-4 or NP-5 may be used to immunize anon-human animal using standard techniques. It may be useful toadminister the antigen in conjunction with an immune adjuvant, as setforth in section 5.3. In cases where a peripheral neuropathy is causedor exacerbated by antibody directed toward an antigen of the invention,animals that produce antibodies against these antigens may produce aperipheral neuropathy comparable to the human condition. In a preferredembodiment of the invention exemplified in Section 12, infra, anon-human animal immunized with sulfatide mixed with either methylatedbovine serum albumin or keyhole limpet hemocyanin (KLH) in completeFreund's adjuvant may serve as a model system for a peripheralneuropathy associated with axonal degeneration and weakness.

Further, the proteins of the invention, namely NP-1, NP-2, NP-3, NP-4and NP-5 may be cloned and characterized using standard molecularbiology techniques. For example, a portion of a protein may besequenced, (e.g. sequence of NP-2 as depicted in FIG. 3) (SEQ ID No: 1)and that amino acid sequence may be used to deduce degenerateoligonucleotide probes that may be used directly to screen genomic orpreferably, cDNA libraries for a clone that contains protein-encodingsequences, or may be used in polymerase chain reaction to amplifyprotein-encoding sequences for subsequent cloning. Once a proteinencoding sequence has been cloned, it may be engineered into anappropriate expression vector so as to enable the production ofrecombinant NP-1, NP-2, NP-3, NP-4, or NP-5 in quantity. Suchrecombinant protein may be used, for example, in the diagnostic: methodsof the invention.

6. EXAMPLE: POLYNEUROPATHY SYNDROME ASSOCIATED WITH SERUM ANTIBODIES TOSULFATIDE AND MYELIN-ASSOCIATED GLYCOPROTEIN 6.1. Materials and Methods6.1.1. Patients

We tested for antibodies to compounds with sulfated carbohydrate(S-carb) moieties in sera from 64 patients in our neuromuscular clinicpopulation who had acquired neuropathies with prominent sensoryinvolvement. Sera from 35 normals and blood bank volunteers, from 21patients with chronic inflammatory demyelinating polyneuropathies (CIDP)with mainly motor involvement and from 20 patients with amyotrophiclateral sclerosis (ALS) were used to establish a range of normal controland disease control values. For each of the 64 sensory neuropathypatients we determined the pattern and degree of sensory and motor loss(Table IV). We also examined electrophysiologic data obtained as part oftheir clinical evaluation. These studies were characterized according toconventional criteria (Nobile-Orazio et al., 1989, Ann. Neurol.26:543-550; Kelly, 1983,Muscle Nerve 6:504-509) as indicative ofpredominantly axonal degeneration, or demyelination, or a mixture ofboth.

6.1.2. Elisa Antibody Assays

Serum was assayed for antibodies to glycolipids and MAG using ELISAmethodology. Glycolipid antigens and chondroitin sulfates were obtainedfrom Sigma (St. Louis, Mo.) . Purified MAG (Quarles, 1988, in "NeuronalAnd Glial Proteins: Structure Function and Clinical Applications"Marangos, Campbell and Cohen, eds. Academic Press, Petaluma, CA, pp.295-320; Quarles et al., 1983, Biochim. Biophys. Acta. 757:140-143) wasa gift from Dr. Richard H. Quarles (NIH). Purified P_(o) protein was agift from Dr. Gihan Tennekoon. Substrates were attached to wells ofmicrotiter plates by two methods (Pestronk et al., 1990, Ann. Neurol.27:316-326). For glycolipids 400 ng in 50 μl of methanol was added towells and evaporated to dryness. Approximately 50 ng MAG orapproximately 200 ng of P_(o) protein and chondroitin sulfate in 100 μlof 0.01M phosphate buffered saline (PBS) pH 7.2 with 0.15M NaCl wereadded to wells and incubated overnight at 4° C. Any remaining bindingsites were blocked with 100 μl of 1% human serum albumin in PBSovernight at 4° C. Plates of MAG but not of g3ycolipids were then washed5 times with 1% bovine serum albumin (BSA) and 0.05% Tween-20 in PBS.

Subsequent steps were performed at 4° C. Between steps washing (×5) wasperformed using PBS with 1% BSA without detergent. All sera were testedin duplicate. Serum was examined by adding 100 μl of dilutions(1:100-1:200,000 in PBS with 1% BSA) to wells for 5 hours (overnight forMAG). The binding of immunoglobulin to glycolipids or MAG was measuredusing overnight (2 hours for MAG) exposure to specific goat anti-humanIgM or IgG linked to horseradish peroxidase (Cappell-Durham, NC) in PBSwith 1% BSA (working dilution 1:20,000). Color was developed by adding100 μl substrate buffer (0.1M citrate buffer pH 4.5 with 0.004% H₂ O₂and 0.1% phenylenediamine) for 20-50 minutes until a standard positivecontrol at a 1:1000 dilution reached an optical density (OD) of 0.6above that of normal controls. OD was then determined for the test andcontrol sera at 450nm. The average OD of normal control sera wassubtracted from the average OD of test sera at each dilution. Titers ofantibodies were calculated from OD data as described in Pestronk et al.(1990, Ann. Neurol. 27:316-326). Readings in the linear range of OD data(0.040 to 0.220 above control) were extrapolated to the value that mightbe expected at a standard dilution of 1:100, multiplied by 1,000 andaveraged. For example, in the test for IgM versus sulfatide in serumfrom patent 6, dilutions of 1:3000 and 1:9000 gave OD readings of 0.150and 0.056 respectively. Using our formula, ##EQU1## we calculated atiter of IgM versus sulfatide of 4,770. In general, a serum with a hightiter of x was detectable (>3 standard deviations (SD) over negativecontrols) in our assays up to a dilution of at least 1/x. We designatedhigh titers as those more than 3 SD above the mean value in our 35patient normal control panel. Our results showed that values ≧900 unitswere high for IgG antibodies against sulfatide and MAG and values ≧1100were high for IgM antibodies against sulfatide and MAG.

6.1.3. Immunoblot Assays

Central nervous system myelin was prepared from human brain (Norton andPoduslo, 1973, J. Neurochem 21:1171-1191). Myelin proteins (100 μg perlane) were fractionated using 12% SDS polyacrylamide gel electrophoresisand transferred onto nitrocellulose sheets (Towbin et al., 1979, Proc.Natl. Acad. Sci. U.S.A. 74:4350-4354). Test sera were diluted1:1000-1:4000 in PBS with 1% BSA and then incubated with nitrocellulosestrips overnight at 4° C. After washing x5 using PBS with 1% BSA thebinding of immunoglobulin was measured using 2-3 hour exposure to goatanti-human IgM linked to horseradish peroxidase in PBS with 1% BSA(working dilution=1:1000). Color was developed with 0.05%diaminobenzidine (DAB) and 0.01% H₂ O₂ in PBS.

6.1.4. Immunostaining after High-Performance Thin-Layer Chromatography(HPTLC)

Sulfatides mixed with a preparation of bovine brain gangliosides (Sigma)were separated by HPTLC on aluminum-backed silica gel 60 HPTLC plates(Merck, Darmstadt, West Germany) using a chloroform: methanol: water(70:30:4) solvent. IgM reactivity in patient sera (1:1000) was detectedby incubation with sera at 4° C. overnight and staining with peroxidaselinked second antibodies and DAB as above.

6.2. Results 6.2.1. Serum Antibody Testing

We performed ELISA testing for antibodies to sulfatide and MAG in seraof 64 patients with peripheral neuropathy syndromes characterized byprominent sensory involvement. Table IV summarizes the findings in 22patients with high titers of antibodies to at least one of the twoantigens. Eighteen patients had high titers of serum antibodies thatreacted with sulfatide. In twelve patients, the high titers ofanti-sulfatide antibodies were IgM and in six patients they were IgG. Inthree of the five patients with the highest titers an IgM paraproteinwas detectable in serum by immunofixation electrophoresis.

Sixteen patients had high titers of anti-MAG antibodies. Thirteen ofthese were IgM class and three were IgG class antibodies. Five of thesix patients with the highest titers had IgM paraproteins.

There appeared to be no correlation between ELISA titers ofantisulfatide and of anti-MAG antibody reactivity in individualpatients. Seven sera (samples 1-7) demonstrated high titer antibodyreactivity only to sulfatide, and four sera (samples 19-22) reacted onlyto MAG. Even the highest titer antibodies to MAG or sulfatide often hadno high titer reactivity to the other antigen. Although there was nocorrelation between titers, half of the sera (11/22) with high levels ofantibody reactivity to one antigen also had high levels to the other.However, three of these sera had IgM reactivity to one antigen but onlyIgG reactivity to the other.

The differential reactivity of the sera that had high ELISA titers onlyto sulfatide or only to MAG was also apparent using overlay methods. Wetested all eleven of these sera (patients 1-7 and 19-22) by Western blotand HPTLC. FIG. 1 shows a comparison of Western blot reactivity ofsample sera with different high titer antibodies as measured by ELISA.The sera with high IgM anti-MAG activity (samples 19-22) stronglystained a protein band (in a CNS myelin protein preparation) thatcorresponds to the molecular weight of MAG (Quarles, 1988, in "Neuronaland Glial Proteins: Structure, Function and Clinical Applications"Marangos Campbell and Cohen eds , Academic Press, Petaluma, Calif., pp.295-320; Quarles et al., 1983, Biochim. Biophys. Acta, 757:140-143).Sera (samples 1-7) with high ELISA anti-sulfatide activity but no ELISAanti-MAG activity did not stain the MAG band. On HPTLC, high titeranti-sulfatide sera (samples 1-7) stained a doublet band correspondingto sulfatide but not the other glycolipids on the plate (FIG. 2).Selective anti-MAG sera (samples 19-22) stained the sulfatide bandweakly or not at all.

We tested several sera in order to determine whether there was arelationship between titers of antibodies to sulfatide and to otherneuropathy-related antigens (Table V). Several antigens were testedincluding: chondroitin sulfate C, a glycosaminoglycan that has beenassociated with axonal sensory-motor neuropathies (Sherman et al., 1983,Neurology 33:192-201; Yee et al., 1989, Acta Neuropathol. 78:57-64);chondroitin sulfate A, another glycosaminoglycan; P_(o), a peripheralmyelin glycoprotein that may react with anti-MAG antibodies (Bollensenet al., 1988, Neurology 38:1266-1270) and GM1 ganglioside and asialo-GM1(GA1), glycolipids that may be associated with motor neuropathies(Nobile-Orazio et al., 1989, Ann. Neurol. 1526:543-550; Pestronk et al.,1990, Ann. Neurol. 27:316-326; Latov, 1987, in "PolyneuropathiesAssociated With Plasma Cell Dyscrasia" Kelly Kyle and Latov eds ,Martinus Nijhoff, Boston, Mass. pp. 51-72; Freddo et al., 1986,Neurology 36:454-458; Steck et al., 1987, Ann. Neurol. 2022:764-767). Wefound that there was no correlation between anti-sulfatide titers andreactivity to chondroitin sulfate A or C, P_(o) protein, GM1-gangliosideor asialo-GM1.

                                      TABLE IV                                    __________________________________________________________________________    Disease                                                                       Duration  Clinical                                                                              Nerve  Antibody titers vs.                                  Age/Sex                                                                            (yrs)                                                                              Syndrome                                                                              Physiology                                                                            Sulfatide                                                                              MAG                                        __________________________________________________________________________     1) 38F                                                                            12   S-Pan   Ax (53; NR)                                                                           1,230                                                                              (IgG)                                                                             --                                          2) 68M                                                                            1    S-Pan; mild M                                                                         Ax (46; 38)                                                                           1,720    --                                          3) 47M                                                                            1    S-Pan   N  (57; 58)                                                                           232,350* --                                          4) 29F                                                                            2    S-Pan; mod M                                                                          M  (33; Nr)                                                                           1,230                                                                              (IgG)                                                                             --                                          5) 44F                                                                            5    S-Pan; mod M                                                                          Ax (56; NR(U))                                                                        902  (IgG)                                                                             --                                          6) 69M                                                                            1    S-Pan   M  (41(P); 30)                                                                        4,770*   --                                          7) 60M                                                                            5    S-Pan; mild M                                                                         M  (50; 30)                                                                           1,365                                                                              (IgG)                                                                             --                                          8) 59M                                                                            1    S-Pan   Ax (45(P); 43)                                                                        2,547    1,232                                                                              (IgG)                                  9) 52F                                                                            1    S-Pan   Ax (40(P); NR)                                                                        7,520    1,936                                      10) 68M                                                                            4    S-Pan; mod M                                                                          D  (31; NR)                                                                           1,920    2,108                                      11) 54M                                                                            3    S-Pan; mild M                                                                         M  (37(P); 39)                                                                        1,392    2,000                                      12) 33M                                                                            4    S-Pan; sev M                                                                          M  (42; NR)                                                                           3,872    3,776                                      13) 75F                                                                            1    S-SF; mild M                                                                          D  (14; NR)                                                                           14,720   1,128                                      14) 44F                                                                            1    S-Pan; mild M                                                                         M  (41; NR)                                                                           2,136    2,360                                      15) 66F                                                                            2    S-Pan; mod M                                                                          M  (37; NR)                                                                           1,904                                                                              (IgG)                                                                             174,000*                                   16) 72M                                                                            5    S-Pan; mild M                                                                         D  (17(U); NR)                                                                        1,206                                                                              (IgG)                                                                             200,000*                                   17) 59M                                                                            5    S-Pan; mild M                                                                         D  (16(U); NR)                                                                        7,848*   4,416*                                     18) 53F                                                                            15   S-Pan; mild M                                                                         D  (43; 34)                                                                           1,054    2,048                                      19) 55F                                                                            9    S-Pan   M  (41; NR)                                                                           --       22,000*                                    20) 61M                                                                            5    S-Pan; mild M                                                                         D  (35; NR)                                                                           --       8,480                                      21) 69M                                                                            10   S-Pan; mod M                                                                          D  (29; NR)                                                                           --       205,056*                                   22) 63M                                                                            4    S-Pan   Ax (54; 52)                                                                           --       1,096                                                                              (IgG)                                 __________________________________________________________________________

Sensory and sensory-motor syndrome patients with high tiers ofantibodies to sulfatide or MAG. Age is at the time of serum testing.

Clinical syndrome: S=sensory; Pan=large and small sensory fibermodalities involved on examination; SF=small fiber sensory modalitiesinvolved; M=motor; sev=severe weakness (3 out of 5 or less) in at leastone muscle group; mod.=moderate weakness (4 out of 5 or worse) in atleast one muscle group; mild=weakness but not worse than 4+ out of 5.

Nerve Physiology: Ax=axonal; M=mixed, moderate features of axon loss anddemyelination; D=demyelination (Kelly, 1983, Muscle Nerve 6:504-509);N=Normal. Numbers in parenthesis: (A;B) A=motor conduction velocity;B=sensory conduction velocity. Unless otherwise noted motor conductionsare from median nerve, sensory values from sural. U=ulnar, P=commonperoneal, N.R.=no response.

Antibody titers: sera with a high titer of x units were generallysignificantly above background at a dilution of x. We have listed allvalues considered high (see methods) for IgM and IgG against sulfatideand MAG. *=monoclonal IgM paraprotein detected by immunofixation.Antibodies were IgM unless noted. -=no high titer antibodies detected.

6.2.2. Correlations between antibody reactivity and clinical andphysiological patterns

Eleven of thirteen patients with high ELISA titers of IgM anti-MAGantibodies (samples 9-21) had a combined sensory plus motor neuropathy(Table IV). Sensory loss usually involved both large and small fibermodalities. Motor findings were often mild but were unequivocallypresent in eleven patients in this group. The distribution ofsensory-motor loss was always greater distally than proximally. Mostoften the signs were symmetric. However, three patients showedconsiderable asymmetry in strength. Nerve conduction studies revealedsome demyelinating features in twelve of the thirteen patients with highIgM anti-MAG antibodies. Seven had predominantly demyelinating changes.Five had mixed demyelinating and axonal abnormalities. Two patients(samples 7 and 8) had high titers of IgG but not IgM anti-MAGantibodies. Both had axonal, sensory polyneuropathies.

In the group of eight patients with high ELISA titers of IgM or IgGanti-sulfatide antibodies but without high titer IgM anti-MAG reactivity(samples 1-8) there were four pure sensory and four sensory plus motorpolyneuropathies. In all these patients sensory loss was distal andinvolved both large and. small fiber modalities. Nerve conductionstudies showed only axonal abnormalities in four patients, mixedfeatures in three and were normal in one. No patient with selectiveanti-sulfatide activity had predominantly demyelinating changes.

None of the sera from 35 normal controls had titers of IgG to MAG orsulfatide ≧900, or titers of IgM to MAG or sulfatide ≧1100.

None of the twelve patients with dorsal root ganglioneuropathy syndromeshad high titers of antibodies to sulfatide or MAG. In other neurologicdisease control groups none of the 20 patients with ALS or the 21 withmotor CIDP had high titers of antibodies to sulfatide or MAG.

6.3. Discussion 6.3.1. Patients with Anti-Sulfatide Antibodies

Our eight patients with high titer serum reactivity to sulfatide,without high titer IgM binding to MAG, had similar clinical syndromes ofpredominantly sensory neuropathy (Table IV). At onset these patientsnoted numbness and paraesthesias or pain in the feet. Symptoms usuallyspread more proximally in the legs and appeared in the hands within ayear of onset. Mild weakness was noted in some patients, but usuallybegan several months to years after the onset of sensory complaints. Onexamination sensory and motor signs were more prominent distally.Reflexes were diminished or absent at the ankles but usually preservedelsewhere. Nerve conduction studies generally showed changes compatiblewith axonal disease but only minor, if any, evidence of demyelination.The incidence of high titers of anti-sulfatide antibodies in a generalpopulation of patients with similar idiopathic axonal sensory-motorneuropathies appears to be at least about 20-30 percent.

6.3.2. Patients With IgM Anti-Mag Antibodies

Sensory symptoms and signs were also a common feature in the anti-MAGneuropathy group (Table IV). However, the patients with high titers ofIgM anti-MAG antibodies differed from the anti-sulfatide group in tworespects.

First, mild to moderate weakness was more common in these patients.Distal weakness was present in 85% (11 of 13) of our patients with highIgM anti-MAG titers. Weakness has also been reported in most previouslydescribed patients with IgM anti-MAG antibodies (Nobile-Orazio et al.,1989, Ann, Neurol. 26:543-550; Steck et al., 1987, Ann. Neurol.22:764-767; Jauberteau et al., 1988, Rev. Neurol. (Paris) 144:474-480;Kelly et al., 1988, Arch. Neurol. 45:1355-1359; Vital et al., 1989, ActaNeuropathol. 79:160-167; Hafler et al., 1986, Neurology 36:75-78).However, only 44% (four out of nine) of the other antibody-positivepatients in our series had weakness.

Second, patients with high titers of IgM anti-MAG antibodies frequentlyhad some physiologic evidence of demyelination (92%; 12 of 13; Table IV)(Nobile-Orazio et 0al., 1989, Ann, Neurol. 26:543-550; Steck et al.,1987, Ann. Neurol. 22:764-767; Jauberteau et al., 1988, Rev. Neurol.(Paris) 144:474-480; Kelly et al., 1988, Arch. Neurol. 45:1355-1359;Vital et al., 1989, Acta Neuropathol. 79:160-167; Hafler et al., 1986,Neurology 36:75-78). A majority (54%; 7 of 13) showed predominantlydemyelinating changes (Kelly, 1983, Muscle Nerve 6:504-509). Incontrast, the patients with only anti-sulfatide antibodies hadpredominantly axonal changes; there was some physiologic evidence ofdemyelination in only 43% (3 of 7) and none had a pattern of predominantdemyelination.

6.3.3. Patients with Anti-S-Carb Antibodies

The results of this study provide evidence that antibodies directedagainst compounds containing S-carb moieties are a frequent feature ofperipheral neuropathies with a prominent sensory component. Thissuggests that compounds containing S-carb may be an antigenic markerthat is particularly abundant on axons or myelin of peripheral sensorynerves. However, the fine specificity of anti-S-carb antibodies seems tovary according to the clinical syndrome. In demyelinating sensory-motorneuropathies, the anti-S-carb antibodies tend to cross react withcompounds containing an SO₄ -3-glucuronic acid as the terminal sugar onthe carbohydrate moiety (Nobile-Orazio et al., 1989, 26:543-550; Latov,1987, in "Polyneuropathies Associated With Plasma Cell Dyscrasia" Kelly,Kyle, Latov, eds , Boston, Martinus Nijhoff, pp. 51-72; Steck et al.,1987, 22:764-767; Bollensen et al., 1988, Neurology 38:1266-1270;Hosokawa et al , 1988, in "Neuroimmunological Diseases" A Igata ed.,Tokyo: University of Tokyo Press, pp. 55-58; Ilyas et al., 1985, Proc.Natl. Acad. Sci. U.S.A. 82:6697-6700). In the patients described herewith predominantly axonal sensory polyneuropathies, anti-sulfatideantibodies may be directed against an epitope that includes an SO₄-3-galactose moiety. Although these sulfated epitopes appear similar,antibodies to one commonly do not cross react well with the other (TableV; Jauberteau et al., 1989, Neuroscience Letters 97:181-184). This wastrue for six of our seven sera with monoclonal proteins and 11 of 22sera overall. The specificity of both types of anti-S-carb antibodies isfurther shown by their lack of general reactivity with other CNSglycolipids or glycoproteins as measured by ELISA, HPTLC andimmunoblotting studies (FIGS. 1, 2; Nobile-Orazio et al., 1989, Ann.Neurol. 26:543-550; Jauberteau et al., 1989, Neuroscience Letters97:181-184).

Others have described patients with anti-MAG antibodies who did not haveserum paraproteins (Nobile-Orazio et al., 1989, Ann. Neurol. 26:543-550;Nobile-Orazio et al., 1984, Neurology 34:218-221); however, reports ofsuch patients are rare. In contrast, only 7 of 22 patients in our serieswith high titers of anti-S-carb antibodies had detectable paraproteins.Thus, the frequency of high titer anti-MAG and anti-sulfatide antibodiesin the absence of a detectable serum M-protein may be greater thanpreviously suspected. Study of sera from other clinically similarpatients with otherwise idiopathic sensory or sensory plus motorneuropathies may uncover high anti-S-carb antibodies directed againstsulfatide or MAG. ELISA assays performed at 4° C., in the absence ofdetergent and using BSA in wash solutions are particularly sensitive forsuch testing (Pestronk et al., 1990, Ann. Neurol. 27:316-326;Marcus etal., 1989, J. Neuroimmunol. 25:255-259). Based on our experience, serumanti-S-carb antibodies are more likely to occur in patients with distalgreater than proximal polyneuropathies than in patients with sensoryganglionopathy with prominent early proximal or upper extremityinvolvement.

                                      TABLE V                                     __________________________________________________________________________    IgM versus                                                                    Patient #                                                                          Sulfatide                                                                           MAG P.sub.o                                                                            Ch--S--A                                                                            Ch--S--C                                                                             GM1                                                                              GA1                                       __________________________________________________________________________     3   232,350                                                                                 0                                                                                0    0     0    0    0                                      13    14,720                                                                              1,128                                                                            1,356                                                                                182   352  265                                                                                465                                     17    7,848                                                                               4,416                                                                              860                                                                              2,732   538  381                                                                              1,068                                     14    2,136                                                                               2,796                                                                            1,280                                                                              2,356 2,336  285                                                                              3,575                                     16      863                                                                              200,000                                                                              0    0     0    0    0                                      __________________________________________________________________________

Patterns of cross reactivity of anti-sulfatide and anti-MAG sera withother sulfated or neuropathy-related antigens. P_(o) =P_(o) protein,Ch-S-A=Chondroitin sulfate A, Ch-S-C=Chondroitin sulfate C, GM1=GM1ganglioside, GA1=asialo-GM1 ganglioside. Titers were measured by ELISA.Note that there is no relation between antibody titers to sulfatide orMAG and those to the other antigens tested.

7. EXAMPLE: CHARACTERIZATION OF NEUROPROTEIN-1 7.1. ProteinIdentification

Neuroprotein-1 (NP-1) is identified by gel chromatography and Westernblotting as 3 protein bands with approximate molecular weights of about36, 38 and 42 kD. The bands migrate between 32 and 47 kD molecularweight markers. NP-1 was specifically identified by its ability to bindto IgM antibodies from 3 sera (numbers W1160, W2333, and W2500). NP-1was enriched in the central nervous system (CNS) and spinal cordnon-myelin white matter. Many sera that react with sulfatide reactedwith this protein, but sera from some neuropathy patients that do notreact with sulfatide may react with NP-1. The 36-42 kD protein bandswere contained in a non-myelin human CNS pellet produced bycentrifugation at 34,000 rpm for 30 minutes in a discontinuous sucrosegradient with layers of 0.32M .and 0.88M sucrose. Further purificationwas obtained after delipidation in ether-ethanol (3:2) and washing threetimes in 1% Triton-X-100 by centrifuging at 10,000 rpm for 10-20minutes. After washing, the pellet from a 20,000 rpm centrifuge spin for30 minutes was separated by 12% polyacrylamide gel electrophoresis(PAGE). The specific protein bands, identified by appropriate molecularweight and antibody binding, were eluted from the gel and used forWestern blotting or ELISA assays. NP-1 was reactive with ricinhemagglutinin and peanut lectin. Thus NP-1 is presumably a glycoproteincontaining terminal Galβ1-3 GalNAc carbohydrate moieties.

7.2. Patient Testing

We have identified over 70 patient sera with high titer (≧1:1000)antibodies to this protein by ELISA testing. These patients generallyhave a mixed axonal and demyelinating sensory-motor polyneuropathy. Serafrom 20 control persons, 21 patients with ALS, and 15 patients withchronic inflammatory demyelinating polyneuropathy (CIDP) did not bind tosulfatide or to NP-1.

8. EXAMPLE: DIFFERENT REACTIVITY OF SERUM IgM TO GM1 GANGLIOSIDE ANDCYCLOPHILIN A IN TREATABLE MULTIFOCAL MOTOR NEUROPATHY 8.1. Materialsand Methods 8.1.1. Patients

We studied patients with motor system disorders or polyneuropathy andhigh serum titers of IgM anti-GM1 antibodies. For this study clinicalsyndromes were assigned to several categories. 1) Seventeen patients hadMMN with distal asymmetric weakness, no definite upper motor neuron orbulbar signs, and motor conduction block on electrodiagnostic testing(Pestronk et al., 1988, Ann. Neurol. 24:73-78); Pestronk et al., 1990,Ann. Neurol. 27:316-326). These were further subdivided into a group of9 patients who improved (with increased strength of at least 1 grade onthe MRC scale) after treatment using cyclophosphamide or chlorambucil.The remaining 8 patients with MMN were either untreated (6 patients) orhad no improvement after immunosuppression (2 patients). 2) Twenty-fivepatients had distal asymmetric LMN signs, no definite evidence of bulbaror upper motor neuron involvement and only axonal changes onelectrodiagnostic testing (Pestronk et al., 1990, 27:316-326) .Thirty-seven patients with classic ALS were defined by previouslyreported criteria used to qualify patients for a series of clinicaltreatment trials (Pestronk et al., 1988, Neurology 38:1457-1461) 4)Forty-one patients had sensory or sensory+motor peripheralpolyneuropathies (PN). 5) Thirty unselected sera from blood bankvolunteers were used to obtain control values.

8.1.2. Antibody Assays

Sera were assayed for antibodies to purified GM1 ganglioside (Sigma) andto the 17 kD neural protein. The protein was purified from a non-myelinpellet (Norton et al., 1973, J. Neurochem. 21:1171-1191) of human CNSwhite matter. The pellet was delipidated with ether-ethanol (3:2),washed in 1% Triton-X-100, again in deionized water and finally in 0.1MTris-HC1, 0.1M PMSF, 5 mM EGTA at pH 7.25. The pellet was thenhomogenized in ice-cold solubilization buffer (25 mM CHAPS, 2M NaCl, 1mM EGTA, 0.15M Na₂ PO₄, 2% glycerol, 0.1M PMSF, pH 7.25), incubated for30 minutes at 4° C. and centrifuged at 100,000 g for 2 hours. Thesupernatant was then desalted, concentrated and subjected to preparativepolyacrylamide gel electrophoresis (PAGE). The specific bands wereidentified (after Western blotting (Pestronk et al., 1991, Neurology41:357-362) of a parallel lane and staining with a human serum (W2393 )that binds strongly to the protein) and eluted from the gel.

Our ELISA methodology has previously been described (Pestronk et al.,1990, Ann. Neurol. 27:316-326; Pestronk, 1990,Muscle & Nerve (in press)"Motor Neuropathies, Motor Neuron Disorders And AntiglycolipidAntibodies"). GM1 ganglioside (400 ng in 50 μl of methanol) was added towells and evaporated to dryness. The purified 17 kD protein (400 ng in100 μl of 0.01M phosphate buffered saline (PBS) pH 7.2 with 0.15 M NaCl)was added to wells and incubated overnight at 4° C. Any remainingbinding sites were blocked overnight at 4° C. with 100 μl of 1% humanserum albumin in PBS for IgM assays and 1% normal goat serum for IgGassays. Plates of the 17 kD protein but not GM1 were then washed fivetimes with 1% bovine serum albumin (BSA) and 0.05% Tween-20 in PBS.Subsequent steps were performed at 4° C. Between steps washing (x 5) wasperformed using PBS with 1% BSA without detergent. Serum was diluted inPBS with 1% BSA and added to wells for 5 hours. Antibody binding to GM1or the 17 kD protein was measured using overnight exposure to specificgoat anti-human IgM or IgG linked to horseradish peroxidase (OrganonTeknika-Cappel, West Chester, PA). Color was developed by addingsubstrate buffer (0.1M citrate buffer, pH 4.5 with 0.004% H₂ O₂ and 0.1%phenylenedramine) until a standard positive control reached an opticaldensity (OD) of 0.6 above normal controls. Titers of antibodies werecalculated from OD data by extrapolating readings to the OD that mightbe expected at a standard dilution of 1:100. In general, a serumantibody with a high titer of x was detectable (>3SD over negativecontrols) up to a dilution of at least 1/x. High titers were more than3SD above the mean of a panel of sera from blood bank volunteers. Values≧350 units were high for serum IgM antibodies against GM1 ganglioside.Values ≧2000 were high for serum IgM antibodies against the 17 kDprotein.

8.1.3. Protein Sequencing

Amino terminal sequencing of proteins was carried out using an AppliedBiosystems Automated Protein Sequencer --Model 477 (Foster City, Calif.)by the Washington University Protein Chemistry Laboratory. Standardreagents and conditions were used.

8.1.4. Western Blot

The non-myelin CNS pellet (10 μg of protein per lane) or purified CyPA(2 μg) were fractionated using 15% PAGE and transferred ontonitrocellulose sheets. Test serums were diluted 1:500-1:4000 in PBS with1% BSA and then incubated with nitrocellulose strips for 2 hours at roomtemperature. After washing x5 using PBS with 1% BSA, the binding ofimmunoglobulin was detected using 1 hour exposure to goat anti-human IgMlinked to horseradish peroxidase in PBS with 1% BSA (working dilution,1:1000). Color was developed with 0.05% diaminobenzidine (DAB) and 0.01%H₂ O₂ in PBS.

8.2. Results 8.2.1. Western Blotting of High Titer Anti-GM1 Sera

We initially tested high titer anti-GM1 sera for binding to neuralproteins by Western blot methodology (FIG. 6). High titer anti-GM1 serawere grouped by patient diagnosis and tested at dilutions of 1:500 ormore. IgM in ALS sera commonly bound selectively to a 17 kD proteinpresent in CNS and peripheral nerve homogenates. Peripheral neuropathysera also often demonstrated binding to the 17 kD protein band. However,the pattern of binding of IgM in neuropathy sera was frequently lessselective than in ALS. Most PN sera reacted with at least one band inaddition to the 17 kD protein. Only 20% of ALS sera showed binding tobands other than the 17 kD protein. IgM reactivity in MMN sera testednever bound to the 17 kD band and only rarely to others in CNS orperipheral nerve homogenates. cl 8.2.2. Characterization of the 17 kDProtein

We attempted to purify the 17 kD protein to quantitate antibody bindingby ELISA and to obtain amino acid sequencing. We found that the 17 kDprotein was concentrated in the non-myelin pellet of CNS white matter.Further purification was obtained by solubilization in high salt (=HAPSbuffer (see methods). Preparative PAGE electrophoresis provided a finalisolation step. Amino terminal sequencing of the 17 kD protein wasobtained after repurification by PAGE and blotting to Immobilon paper.The sequence of the first 30 N-terminal amino acids (SEQ ID NO: 1 ) wasvirtually identical to that of cyclophilin A (FIG. 3), SEQ ID NO: 2 a17-18 kD peptidyl-prolyl cis-trans-isomerase with cyclosporine bindingproperties (Haendler et al., 1987, EMBO J. 6:947-950).

8.2.3. Elisa Measurement of Serum IgM Reactivity to theCyclophilin-A-Like Protein (CyPA)

We used ELISA methodology to measure IGM antibody titers to CyPA in serawith high titers of IgM anti-GM1 antibodies. In these sera the highesttiters of anti-GM1 antibodies (>7000) were generally in patients withMMN or LMN disorders (FIG. 7). There were significantly more (p<0.01)patients with anti-GM1 titers above 7000 in these groups than in ALS orPN groups.. However, there was considerable overlap in titers among thediagnostic groups.

In a panel of 30 unselected control sera from blood bank volunteers themean titer of IgM against CyPA was 243+468 (standard deviation) units,with the mean plus 3 standard deviations (SD) at 1647 units. In patientsera with high anti-GM1 titers, anti-CyPA titers varied from 0 to 75,160(FIG. 8). There were significantly (p<0.001) more high titer sera in theALS (81%; 30/37) and PN groups (79%; 33/42) than in the MMN group (30%;5/17).

The ratio of IgM titers to CyPA and GM1 for each serum (CyPA:GM1 ratio)provided the best distinction between MMN and other patient groups (FIG.9). The median CyPA:GM1 ratio for all sera tested was 2.37. Inindividual sera CyPA:GM1 ratios varied greatly, from 0 to 47. There wasno correlation between the absolute titer of IgM anti-GM1 antibodies ina serum and the CyPA:GM1 ratio. Patients with treatable MMN all had lowratios, ranging from 0 to 0.78. Overall, 82% (14/17) of MMN patients hadratios below 0.79 (median=0.12). Patients with ALS and polyneuropathyhad significantly ((p<0.0001) higher CyPA:GM1 ratios. The median ratiofor ALS sera was 3.38 with only 14% (5/37) below 0.79. The median ratiofor polyneuropathy sera was 3.02 with only 7% (3/41) below 0.79. Theoverall statistics for the LMN group were intermediate with a medianratio of 0.64 and 52% (13/25) below 0.79. The intermediate value for theLMN group resulted from a large number of CyPA:GM1 ratios with a valueof 0 (36%; 9/25). If sera with ratios of 0 were deleted from the LMN andALS groups, then the remaining populations of values were notsignificantly different.

8.3. Discussion 8.3.1. Fine Specificities of Anti-GM1 Antibodies

Sera with high titers of antibodies to GM1 ganglioside may also reactwith other glycolipids or glycoproteins (Freddo et al., 1986, Neurology3,6:454-459; Pestronk et al., 1990, Ann. Neurol. 27:316-326; Shy et al.,1989, Ann. Neurol. 25:511-513; Latov et al., 1988, Neurology 38:763-768;Baba et al., 1989, J. Neuroimmunol. 25:143-150; Kusunoki et al , 1989 J.Neuroimmunol 21:177-181; Nardelli et al., 1988, Ann. Neurol.23:524-528). The patterns of serum reactivity depend in part oninteractions of the antibodies with specific epitopes on thecarbohydrate or lipid moieties of GM1. Antibodies that react with theterminal disaccharide on GM1, Galβ1-3Ga1NAc, often cross react withother glycolipids that contain the same disaccharide, includingasialo-GM1 and GD1b gangliosides. Antibodies with binding propertiesthat involve the lipid moiety on GM1 may react well with a wide spectrumof other glycolipids, but only poorly with glycoproteins (Chaudhry etal., 1990, Neurology 40:118S).

There is some data regarding the association of particular serum bindingpatterns with specific clinical syndromes. In MMN and LMN syndromes 3major fine specificities of anti-GM1 antibodies have been defined(Pestronk et al., 1990, Ann. Neurol. 27:316-326; Sadiq et al., 1990,Neurology 40:1067-1072; Baba et al., 1989, J. Neuroimmunol. 25:143-150).Each of these reacts with precise carbohydrate epitopes on GM1. Changesin the terminal galactose of GM1, such as addition of a sialic acid,greatly reduce the binding of anti-GM1 antibodies from motor neuropathyand LMN patients. In contrast, the binding of antibodies that ariseafter immunization with GM1 is less affected by changes in thecarbohydrate moiety (Chaudhry et al., 1990, Neurology 40:118S).

The environment of the GM1 molecule variably influences the binding ofanti-GM1 antibodies in different disorders. Anti-GM1 antibodies frompatients with ALS generally bind well to GM1 in a lipid, membrane-likeenvironment. However, antibodies from patients with MMN and INNsyndromes often do not react with GM1 in membranes.

Despite the correlations between antibody specificity and clinicalsyndromes, the diagnostic and pathogenic role of anti-GM1 antibodiesrequire further investigation. There is evidence that some anti-GM1antibodies can bind to neural structures including motor neuron cellbodies and nerve terminals (Schluep et al., 1988, Neurology38:1890-1892; Thomas et al., 1989, J. Neuroimmunol. 23:167-174; Thomaset al., 1990, J. Neuropath Exp. Neurol. 49:89-95). Immunization ofrabbits with GM1 ganglioside may induce neuropathy, possibly with motorconduction block (Nagai et al., 1976, Neurosci. Lett. 2:107-111; Thomas,et al., 1990, Ann. Neurology 28:238). However, it is important toexplain why a range of neuropathy and motor neuron syndromes areassociated with anti-GM1 antibody reactivity. Overlap in antibodybinding patterns between diagnostic groups also limits the diagnosticutility of anti-GM1 antibody testing.

8.3.2. Reactivity of Anti-GM1 Sera With CyPA

The results of our Western blot and ELISA testing show that the patternof serum IgM reactivity in MMN often differs from the patterns in ALSand polyneuropathy. Serum IgM from MMN patients generally reactsconsiderably more strongly with GM1 ganglioside than with CyPA. Many MMNsera do not react with CyPA at all. Most (82%) have CyPA:GM1 ratios ofless than 0.79. In contrast there is commonly high titer IgM reactivityto CyPA in anti-GM1 sera from other patient groups. Some ALS andpolyneuropathy sera react to CyPA in titers that are 10 to 40 timesgreater than those to GM1. Few (10%) have CyPA:GM1 ratios less than0.79. Thus, measurement of CyPA GM1 ratios in high titer anti-GM1 seracan increase specificity for MMN 10-fold. A low CyPA:GM1 ratio (<0.79)occurs in most patients with MMN, but 90% of anti-GM1 sera from otherdisorders have high ratios. Further studies are necessary to determinewhether the patterns of IgM binding result from cross reactivity ofindividual IgM antibodies with both CyPA and GM1, or from the binding ofdifferent IgM molecules in the same serum.

8.3.3. Implications of Anti-CyPA Reactivity in Anti-GM1 Sera

CyPA is a member of a conserved class of proteins that bind theimmunosuppressive drug cyclosporin A (Handschumacher et al., 1984,Science 226:544-547; Koletsky et al., 1986, J. Immunol. 137:1054-1059;Hohman et al., 1990, The New Biologist 2:663-672; Haendler et al., 1987,EMBO J. 6:947-950; Haendler et al., 1990, Eur. J. Biochem. 190:477-482;Iwai et al., 1990, Kidney Internatl. 37:1460-1465; Price et al., 1991,Proc. Natl. Acad. Sci. 88:1903-1907; Fairley, 1990, J. Am. Acad.Dermatol. 23:1329-1334). The degree of binding of most, but not all,cyclosporin A derivatives to CyPA correlates with theirimmunosuppressive potential (Quesniaux et al., 1987, Eur. J. Immunol.17:1359-1365; Donnelly et al., 1991, Clinical Biochem. 24:71-74; Sigalet al., 1991, J. Exp. Med. 173:619-628). In most species cyclophilinsare peptidyl-prolyl cis-trans isomerase enzymes that catalyze proteinfolding. The major cytosolic binding protein for anotherimmunosuppressant, FK-506, has a similar enzymatic activity (Siekierkaet al., 1989, J. Immunol. 143:1580-1583; Harding et al., 1989, Nature(Lond.) 341:758-760). A cyclophilin-like molecule is involved in asignal transduction pathway (Schneuwly et al., 1989, Proc. Natl. Acad.Sci. 86:5390-5394; Shieh et al., 1989, Nature (Lond.) 338:67-70).Cyclophilins are found in many cell types but are especially abundant inneural tissue (Koletsky et al., 1986, J. Immunol. 137:1054-1059; Lad etal., 1991, Molec. Brain Res. 9:239-244; Ryffel et al., 1991, Immunol.72:399-404).

The reactivity of anti-GM1 sera with CyPA is another example of naturalautoantibodies directed against intracellular components (S. Avrameas,1991, Immunol. Today 12:154-159). Low titers of autoantibodies arenonspecific and may occur in normals as well as a variety of diseasestates. High tiers of antibodies against intracellular components havebeen associated with specific autoimmune disorders, includinginflammatory myopathies (Plotz et al., 1989, Annals of Intern. Med.111:143-157), lupus syndromes and paracarcinomatous sensoryneuronopathies (Dalmau et al., 1990, Ann. Neurol. 27:544-552).

Some autoantibodies can enter intracellular compartments of the lowermotor neuron (Yamamoto et al., 1987,. Neurology 37:843-846; Fishman etal., 1989, Neurology 39(suppl 1):402; Fabian, 1988, Neurology38:1775-1780; Engelhardt et al., 1990, Arch. Neurol. 47:1210-1216).Intracellular antibodies could bind to CyPA and inhibit its function.Binding to CyPA in renal cells has been associated with nephrotoxicity(Ryffel, et al., 1991, Immunol. 72:399-404). However, the pathogenicityof antibodies to intracellular components has been difficult to define.Most attempts to study the effects of these antibodies involveimmunization of animals with the target antigen or passive transfer ofappropriate serum. Our data suggest that definition of anti-GM1 serumspecificities, and cross-reactivity with proteins such as CyPA, isnecessary before such results can be interpreted. For MMN autoantibodieswith strong specificity for carbohydrate moieties on GM1 but with littlereactivity to CyPA should be studies. For definition of the relation ofanti-CyPA antibodies to ALS and peripheral neuropathy, it will beimportant to determine differences in serum reactivity between the twosyndromes. Identification of an antibody binding pattern with somespecificity for ALS would likely provide a clue to the mechanismunderlying the disorder.

8.3.4. Diagnostic Testing

The primary reason for the clinical measurement of anti-GM1 antibodiesis as a diagnostic aid in identifying MMN. This disorder is probablyimmune-mediated and treatable (Pestronk et al., 1988, Ann. Neurol24:73-78; Pestronk et al., 1990, Ann. Neurol. 27:316-326). MMN has atherapeutic response profile of considerable improvement (in 8.0% ofpatients) in strength after treatment with sufficient doses ofcyclophosphamide. Prednisone is generally not effective.

High titers of anti-GM1 antibodies occur in 60-80% 80% of patients withMMN (Pestronk et al., 1990, Ann. Neurol. 27:316-326). However, thediagnostic utility of anti-GM1 testing is limited by the occurrence ofthese antibodies in 10-15% of patients with more common disorders,including ALS and polyneuropathy. Our data now show that combinedtesting for antibodies to GM1 and to CyPA provides a 5-10-fold increasein specificity with little reduction in sensitivity. Low CyPA:GM1antibody ratios are present in most MMN patients and in all those whohave responded to cyclophosphamide but not to prednisone treatment. Themeaning of low CyPA:GM1 ;ratios in other disorders with high titers ofanti-GM1 antibodies remains to be determined. It will be especiallyinteresting to compare the response to different immunosuppressiveregimens in LMN and neuropathy syndromes with high or low CyPA:GM1ratios.

9. EXAMPLE: CHARACTERIZATION OF NEUROPROTEIN-3 9.1. ProteinIdentification

Neuroprotein-3 (NP-3) has a molecular weight of 50-54 kD. It migrates on12% PAGE just above the location of the Wolfgram proteins in aseparation of human white matter or myelin. NP-3 is enriched in CNSmyelin. It is specifically identified by the binding of serum W1763.Purification was achieved from myelin prepared by the method of Nortonand Poduslo, 1.973, J. Neurochem. 21:1171-191. CNS myelin wasdelipidated using a mixture of ether and ethanol at a ratio of 3:2,washed with 1% Triton-X-100. Pellets after each of these washes wereobtained by centrifuging at 10,000 rpm for 10-20 minutes. The finalpellet was isolated, dissolved in 2 percent SDS, and then subjected topreparative electrophoresis on 12% PAGE. The specific protein waslocated on the gel by Western blotting with serum W1763 and molecularweight identification. The NP-3 band was then eluted from the PAGE geland concentrated.

Data suggests that NP-3 may be highly homologous or identical tobeta-tubulin. The first 24 amino acid residues of NP-3, depicted in FIG.4, (SEQ ID NO: 3) are strongly homologous with beta-tubulin (SEQ ID NO:4), and serum W1763, which binds to NP-3, binds to beta-tubulin.Further, monoclonal antibodies raised against NP-3 react withbeta-tubulin.

9.2. Patient Testing

Patient sera were tested for antibodies against NP-3 by Western blottingand ELISA methodology. Normal values for levels of antibodies againstNP-3 are less than 1:1000. We have tested over 60 sera from patientswith inflammatory demyelinating polyneuropathies includingGuillain-Barre and CIDP. Our results show that 40% of patients withthese disorders have IgM or IgG antibodies in a high titer against NP-3.Testing of the same sera against other glycolipids and glycoproteinsincluding GM1, sulfatide and panels of neutral and acidic glycolipids inMAG show that less than 5-10% have high titers of serum antibodiesagainst other antigenic targets. Antibodies are present in high titer atthe onset of Guillain-Barre syndrome and fall over the course of thedisease.

10. EXAMPLE: NEUROPROTEIN-4

Neuroprotein-4 (NP-4) has a molecular weight of approximately 20-24 kD.It migrates just above the large basic: protein band on 15% PAGE,. Theprotein is identified by binding with serum W1945. It was prepared bythe method as set forth for NP-2 (supra), and including the washing inTris buffer. The pellet was then dissolved in 2 percent SDS andsubjected to PAGE on a 15 percent polyacrylamide gel. A protein bandhaving a molecular weight of about 20-24 was identified. Western blotsof serum from 14 patients with polyneuropathies show that 5 patientshave serum titers of 1:500 or higher to NP-4.

11. EXAMPLE: NEUROPROTEIN-5

Neuroprotein-5 (NP-5) has a molecular weight of approximately 30-32 kD.It was prepared by differential centrifugation, washing and elution ofspecific 30-32 kD bands from PAGE gels by methods similar to thoseutilized in the preparation of NP-4. It is identified by the binding ofserum 1.0286. NP-5 is present in peripheral nerve and non-myelin brainwhite matter.

12. EXAMPLE: A MODEL OF DISEASE PRODUCTION BY INDUCING ANTI-SULFATIDEANTIBODIES IN EXPERIMENTAL ANIMALS

Eight guinea pigs were immunized with 0.5 mg of sulfatide mixed witheither methylated bovine serum albumin or KLH in complete Freund'sadjuvant. One month later the animals were reimmunized with a similarmixture and incomplete Freunds adjuvant. One to two weeks after thereimmunization 3 animals developed significant weakness. The illness wasterminal in two. Pathological studies show mild evidence of axonaldegeneration in peripheral nerves. Control animals immunized withmethylated BSA or KLH alone did not become ill.

13. EXAMPLE: MONOCLONAL ANTIBODIES TO NEUROPROTEIN-1

DA x Lewis hybrid F1 generation rats were immunized with NP-1 togetherwith Freund's adjuvant and hybridomas were produced using standardtechniques.

Four monoclonal antibodies, A1A1.6, A2H3.7, A2H10.1 and A5H10.1 havebeen produced. By Western blot each of these antibodies reacts with the3 bands in the NP-1 triplet (36 kD, 38 kD and. 42 kD) plus a 30 kDdoublet. This suggests that the NP-1 protein bands are comprised of asingle protein with different post-translational modifications. Byimmunocytochemistry these antibodies stain fibrillary cellular processesin the central nervous system that are especially abundant in spinalgrey matter and cortex.

14. EXAMPLE MONOCLONAL ANTIBODIES TO NEUROPROTEIN-2

DA x Lewis hybrid F1 generation rats were immunized with NP-2 togetherwith Freund's adjuvant and hybridomas were produced using standardtechniques.

Four monoclonal antibodies, B3H12, B5G10, B5G12, B5H10 have beenproduced. B5H10 reacts selectively to NP-2 in a pattern similar to theoriginal W2393 test serum. In immunocytochemistry of neural tissue theB5H10 antibody binds to cells (possibly their nuclei) in peripheralnerve and the cerebellum (especially in the granular layer). B3H12reacts weakly to NP-2 on Western blot. At high dilutions (1:250) itbinds selectively to a 22 kD protein in the non-myelin pellet from humanbrain. In neural tissue the B3H12 antibody binds to cellular processes.In peripheral nerve axons are strongly stained. In the cerebellumprocesses surrounding Purkinge cells are selectively stained. B5G12reacts equally with NP-2 and a 22 kD protein by Western blottingmethods. It also binds to a smaller 10-12 kD protein. B5G10 reactsweakly with NP-2 on Western blot. It binds strongly to a 46-50 kDprotein in non-myelin fractions of human CNS. It binds best to anapproximately 55-65 kD protein in peripheral nerve.

15. EXAMPLE: MONOCLONAL ANTIBODIES TO NEUROPROTEIN-3

DA x Lewis F1 generation rats were immunized with NP-3 together withFreund's adjuvant and hybridomas were produced using standardtechniques.

Four monoclonal antibodies, (C1F10, C2F3, C1H3, C2H1) to NP-3 have beenproduced. By ELISA they react strongly with NP-3 and with tubulinextracted from the myelin pellet from human brain. They cross react tovarying degrees with purified bovine brain tubulin.

Various publications have been cited herein that are incorporated byreference in their entirety.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       Xaa AsnProThrValPhePheAspIleAlaValAspGlyGluProLeu                             151015                                                                        GlyLysValXaaPheGluLeuPheAlaAspLys                                             20 25                                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValAsnProThrValPhePheAspIleAlaValA spGlyGluPro                             151015                                                                        LeuGlyArgValSerPheGluLeuPheAlaAspLys                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetArgGluIleValSerIleGlnAlaGlyGlnAlaGlyAsnGlnIle                              15 1015                                                                       GlyAlaLysPheXaaGluValIle                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetArgGluIleValHisValGlnAlaGlyGlnCysGlyAsnGlnIle                              151015                                                                        GlyAlaLysPheTrpGluValIle                                                       20                                                                       

What is claimed is:
 1. A method aiding in the diagnosis of a peripheralneuropathy in a patient comprising:reacting a serum sample from thepatient with sulfatide; and determining the titer of antisulfatide IgGand IgM antibody in the serum sample that binds to sulfatide, in which atiter greater than about 1:900 correlates positively with apredominantly axonal neuropathy.
 2. The method of claim 1 in which thepredominantly axonal neuropathy is predominantly sensory.
 3. The methodof claim 2 in which the neuropathy has a clinical history of presentingfirst as numbness and paresthesias or pain in the feet, and thenspreading more proximately in the legs and eventually involving thehands, then the arms.
 4. The method of claim 1 which is a pure sensoryneuropathy.
 5. The method of claim 1 which is a sensory motorneuropathy.
 6. A method aiding in the diagnosis of a peripheralneuropathy in a patient comprising: reacting a serum sample from thepatient with sulfatide; and determining the titer of anti-sulfatide IgGand IgM antibody in the serum sample that binds to sulfatide, in which atiter greater than about 1:1100 correlates positively with apredominantly axonal neuropathy.
 7. The method of claim 6 in which thepredominantly axonal neuropathy is predominantly sensory.
 8. The methodof claim 7 in which the neuropathy has a clinical history of presentingfirst as numbness and paresthesias or pain in the feet, and thenspreading more proximately in the legs and eventually involving thehands, then the arms.
 9. The method of claim 6 which is a pure sensoryneuropathy.
 10. The method of claim 6 which is a sensory motorneuropathy.